1.4 The dynamic nature of the podosomes of actin rings.............................. 25

1.5 In unactivated osteoclasts, V-ATPase is not present at the plasma
membrane but is stored in cytoplasmic vesicles; but upon activation, it is
transported via actin filaments to the ruffled membrane. .............. ........... 26

4.4 siRNA 120649, but not a control siRNA (120653), effectively knocks
down the cortactin content to an undetectable level of osteoclast-like cell
extract after 30 hours compared with actin ........................................... 91

4.6 An siRNA known to downregulate cortactin (Ambion) effectively knocks
down the cortactin content of osteoclast-like cell extract to an
undetectable level after 30 hours compared with actin ........................... 93

movement can be accelerated by the removal of OPG. Compared to wild type

OPG littermates, OPG knock out mice show increased osteoclast number and

increased alveolar bone resorption (127). In the future, the power of the

osteoclast may be able to be harnessed to enhance the treatment of the dental

patient.

General Purpose of Research

The general purpose of the work presented in this dissertation has been to

learn more about the actin ring of osteoclasts, its characteristics and composition

and requirements for formation. In addition, we sought to identify a relationship

between components of the actin ring and V-ATPase, another specialized

structure of the osteoclast.

Figure 1.1. Resorbing osteoclast. Once the osteoclast attaches to bone, there is
segregation of an extracellular compartment between it and the bony surface.
The area of tight adhesion segregating this extracellular compartment is termed
the sealing zone. Bounded by the sealing zone is the ruffled membrane. The
ruffled membrane is a convoluted membrane packed with vacuolar proton
ATPase (VATPase), the osteoclast proton pump (3). Bone degradation is
initiated by hydration of carbon dioxide to carbonic acid by carbonic anhydrase II
(CA II). The carbonic acid then dissociates into protons and bicarbonate ions. At
the apical membrane, the protons are pumped into the extracellular compartment
via the V-ATPase. At the basolateral membrane, bicarbonate is exchanged for
chloride ions in an energy dependent manner. The chloride ions, which have
entered the osteoclast, pass into the extracellular compartment through an anion
channel coupled to the V-ATPase. The protons and chloride ions form
hydrochloric acid and reduce the pH in the extracellular compartment to
approximately 4.5, which allows the demineralization of the bone mineral and
exposes the organic matrix of the bone. Cathepsin K, an acid cysteine
proteinase, is then able to degrade the bone matrix. The degraded products,
collagen and calcium, are then transcytosed through the osteoclast and secreted
into the microenvironment through the basolateral membrane. (Teitelbaum et al.
J Bone Miner Res 2000; 18:344-349) (3)

I Immuse System

Y RANK
SRANKL

re nbrtle I
0F1

Pmahtinr of Fedef Or1Kte0ea
iemniCaleipa Siem CeA Prhcrr
(CFll-C tl

M-CSF

M-CSF
T7VFI
ILI

Orot i Sutnrl
amd ardsklm
A

TNFt N7VFa
ILI ILl

Figure 1.2. The OPG/RANK/RANKL triad plays an important role in the bone,
immune, and vascular systems. In the bone system, the interaction between
OPG and RANKL promotes either osteoclast differentiation and survival or
osteoclast apoptosis. (Theoleyre et al. Cytokine and Growth Factor Reviews.
2004; 15:457-475) (17)

Vascular System a
Ewdhri sad sm moth mmsc lt l

* m

96d W

Osteoblastic
Stromal Cells

,*I --.. Pi3K
.. ..... Other kiases ?

.........; -KT
..IKK KKK 6, o MEKI AKT'

Differentiation

Osteoclastic Survival
Cystoskeletal effects

Figure 1.3. Binding of the adaptor protein TRAF6 is the initial step in RANKL
signaling. Down stream targets of TRAF6 include nuclear transcription factors,
such as NFKB, and signal transduction molecules, such as c-Src. (Theoleyre et
al. Cytokine and Growth Factor Reviews. 2004; 15:457-475) (17)

LATRUNCULIN A

* .

Figure 1.4. The dynamic nature of the podosomes of actin rings. Rhodamine
actin was incorporated into saponin permeabilized osteoclast like cells. In the
control cells, the rhodamine actin was quickly incorporated (within 10 minutes)
into the actin rings of osteoclasts. In the latrunculin A treated cells, which inhibits
G-actin from polymerization, a complete loss of the actin ring was observed.
(Hurst and Holliday, unpublished)

CONTROL

,~8~+,

V-ATPase

Figure 1.5. In unactivated osteoclasts, V-ATPase is not present at the plasma
membrane but is stored in cytoplasmic vesicles, but upon activation, it is
transported via actin filaments to the ruffled membrane. Mouse marrow
osteoclasts were loaded onto bovine cortical bone slices cultured for 2 days, and
fixed and stained with anti-V-ATPase antibody and phalloidin. This micrograph is
representative of an early resorptive osteoclast. The white arrow identifies a
region where the V-ATPase has been transported to the ruffled membrane which
is bounded by actin. The black arrow, below, identifies a unactivated region,
where the V-ATPase and actin are still found to be co-localized in cytoplasmic
vesicles. (Lee et al. J Biol Chem. 1999; 274(41):29164-29171) (9)

the gelsolin "knockout" mouse is mildly osteopetrotic, suggesting a role for

gelsolin in bone resorption (165). However, the mildness of the osteopetrosis

suggests other mechanisms contribute to the cytoskeletal dynamics required for

bone resorption (166, 167). A strong possibility may be coordination between

gelsolin and the Arp2/3 complex. A recent model describing podosomes

suggests a balance of actin polymerization, which, based on our results, is likely

regulated by the Arp2/3 complex, and filament cleavage, by proteins like gelsolin

(33). This balance could account for the structure and dynamics of podosomes.

In summary, the Arp2/3 complex is present in the podosomal structures of

the actin rings of osteoclasts. Knockdown of Arp2 using siRNA shows that the

Arp2/3 complex is required for actin ring formation. These data suggest that the

Arp2/3 complex plays a role in osteoclastic bone resorption and may provide a

target for therapeutic agents designed to limit the activity of osteoclasts.

Inactive Active
"Open" "-Cksed"

Figure 2.1. The Arp 2/3 complex. A) Crystal structure of the 7 subunits of the
Arp2/3 complex. B) The Arp2/3 complex remains in an inactive conformation.
Upon activation by WASP family members, the Arp2 and Arp3 subunits undergo
a conformational change and allow the complex to become active and participate
in actin polymerization. (Robinson et al. Science. 2001; 294:1679-1684) (138)

Figure 2.2. The purification of the Arp2/3 complex from human platelets. The
Arp2/3 complex was purified from human platelets by a previously published
method by Welch and Mitchison using conventional chromatography. Each lane
depicts the elution from the columns run with purified Arp2/3 complex obtained
after gel filtration.

~L_ --

11 Mr --At

sc
f"- '~

A Arp3 Arp2

C
B I

I
(,
z

w

z :

anti-actin anti-Arp2 anti-Arp3

Figure 2.3. Arp2 and Arp3 are upregulated during osteoclastogenesis. (A)
Human platelet Arp2/3 complex was subjected to SDS-PAGE, blotted to
nitrocellulose, and probed with antibodies against Arp3 and Arp2, and the bound
antibody was detected by chemiluminescence. B) RAW 264.7 cells were
cultures with (black bars) or without (white bars) RANKL. Total protein was
extracted and equal amounts of protein were loaded and separated by SDS-
PAGE and transferred to nitrocellulose and probed with anti-actin, anti-Arp2 and
anti-Arp3 antibodies. Arp2 and Arp3 expression was upregulated during
osteoclastogenesis compared with actin. C) Quantitation of four independent
blots confirmed upregulation of Arp2 and Arp3 as osteoclasts differentiated.
Error bars represent standard error. p < 0.05 by student's t-test.

35000

30000

25000

20000

15000

10000

5000

ArP3

Arp2

Arp3b Arp3 GAPDH

-

S U S U S U
Figure 2.4. The two isoforms of Arp3, Arp3 and Arp3-beta, are present in
unactivated and activated osteoclasts. RAW 264.7 cells were cultured with
(stimulated) or without (unstimulated) RANKL. Cells were harvested and RNA
was obtained using RNAeasy Mini Kit (Qiagen, Valencia, CA). RT-PCR was
performed using primers specific to Arp3 and Arp3-beta. Both Arp3 and Arp3-
beta were present and are upregulated in response to RANKL stimulation.

Figure 2.5. Arp2/3 complex is present in the actin rings of osteoclasts. Mouse
marrow osteoclasts were loaded onto bovine cortical bone slices (A-C) or glass
coverslips (D-E), cultured for 2 days, and fixed and stained with anti-Arp3
antibody (A and D) and phalloidin (B and E). Images were merged (C and F),
with Arp3 staining pseudocolored green and phalloidin pseudocolored red. Co-
localization of the two is yellow. A-C) A projection of 15 confocal slices (0.5 pm)
is shown. The arrow indicated the actin ring. The green staining of the nuclei
was the result of cross reactivity by the secondary antibody. Note the yellow
staining of the actin ring in the merged image indicating co-localization. D-F)
This is an image of a single optical section (0.5 pm) of a mouse marrow
osteoclast on a glass coverslip. The small arrow points to Arp2/3-rich spots; the
large arrow identifies the actin rings. The size bar is equivalent to 5 |tm in A-C
and 25 pm in D-F.

H I -

Figure 2.6. Arp2/3 complex is enriched relative to F-actin near the sealing zone.
A and B) A projection of the edge of an osteoclast on a coverslip is shown,
stained with (A) anti-Arp3 or (B) phalloidin. C-E) The images in A and B were
computer rotated 900 to examine the cell in side view. The apical side is down.
The podosomal nature of the ring is readily apparent. As shown by the arrows,
Arp3 (pseudocolored green) was enriched near the apical surface (the contact
area with the coverslip), whereas microfilaments (pseudocolored red) were
enriched at the basolateral boundary of the actin ring. Areas of co-localization
are yellow. F and G) The image of a resorbing osteoclast on a bone slice is
shown. H and I) A section of the actin ring is identified from F and G. J) The
images in H and I were then merged and rotated 900 so that the apical surface
was down. Arp3 is pseudocolored green and phalloidin is red. As observed in
the osteoclast on a glass coverslip, Arp3 is enriched near the apical boundary
near the sealing zone (arrow). The size bar is 10 am in A and B; 5 [m in C-l,
and 2 am in J.

Figure 2.7. Arp2/3 does not co-localize with vinculin in actin rings. RAW 264.7
cells were stimulated with RANKL to differentiate into osteoclast-like cells and
fixed and stained with either anti-Arp3 or anti-vinculin. The images were merged.
A) Image of actin ring stained with anti-Arp3 and pseudocolored red. B) Image
of actin ring stained with anti-vinculin and pseudocolored green. C) Merged
image of A and B. Note there is little co-localization between Arp3 and vinculin.
The size bar is 3 tm.

Actin

Control

Cytochalasin D

Echistatin

Wortmannin

Figure 2.8. Treatment with the chemical agents, cytochalasin D, echistatin and
wortmannin, cause a disruption of the actin rings of osteoclasts. Mouse marrow
osteoclasts were loaded onto bovine cortical bone slices or glass coverslips,
cultured for 2 days, and either untreated or treated with with cytochalasin D,
echistatin or wortmannin for 30 minutes and fixed and stained with anti-Arp3
antibody and phalloidin. Note the disruption of the actin ring in all cells but co-
localization of the Arp2/3 complex with actin remains stable.

Arp3

MERGE

Figure 2.9. Arp2/3 remains co-localized in the actin based podosomal core
regardless of actin ring disruption by wortmannin. RAW 264.7 cells were
cultured with RANKL until osteoclast-like cells were observed. The cells were
then treated with 100 nM wortmannin for 15 mintues, after which they were fixed
and stained with either rhodamine phalloidin or anti-Arp3 antibody. Although actin
ring structure has been disrupted, Arp3 continues to co-localize with actin in the
podosomal core.

160

JI I 7-

Control Wortmannin

Echistatin

Figure 2.10. Wortmannin and echistatin treatment of osteoclasts results in a
decrease in the number of actin rings. Actin rings were counted after either no
treatment or treatment with wortmannin or echistatin. A significant decrease in
actin rings, more than 90%, was observed after treatment with either inhibitor.

ARP3

ACTIN

V -'.-l

NO
TREATMENT

19944

19942

I "- I

I.- m

Figure 2.11. siRNA 19942 but not 19944 reduces the Arp2 content of osteoclast-
like cell extract 70% after 30 hours compared with actin. RAW 264.7 cells were
stimulated with RANKL. Just as large, multinucleated osteoclasts began to
appear, cells were transfected as noted. Cells transfected with siRNA 19942,
which had proved effective at knocking down Arp2 in preliminary experiments,
reduced Arp2 levels dramatically compared with either control cells or cells
transfected with an ineffective siRNA 19944.

ARP2

ACTIN

TRITC-PHALLOIDIN

-NO TREATMENT

19941

19942

Figure 2.12. Actin rings are disrupted in Arp2 knockdown. Untransfected RAW
264.7 osteoclast-like cells or osteoclast-like cells transfected with ineffective
siRNA (19941) or effective siRNA (19942) were fixed after 30 hours and
examined for the presence of fluorescent oligo marker of transfection (left panels)
or F-actin by staining with phalloidin (right panels). The photographs are
representative cells. The effective siRNA disrupted the ability of the osteoclasts
to form actin rings. The size bar equals 25 pm.

FITC-OLIGOMER

Figure 2.13. Actin rings are disrupted in marrow osteoclasts on coverslips or on
bone slices by siRNA directed against Arp2. Mouse marrow in tissue culture
plates was stimulated with calcitriol for 5 days to produce osteoclasts. These
were scraped and loaded onto coverslips (A-D) or bone slices (E-H) and
transfected with (A, B, G, and H) 19942 or (C-F) 19941. The cells were stained
with phalloidin (B, D, E, and G) or the fluorescent oligomer (A, C, F, and H) was
detected. Note that in osteoclasts transfected with the effective siRNA (19942),
no actin rings were present. In cells transfected with the ineffective control
siRNA (19941), actin rings appeared normal. Standard bar in D is for A-D and
represents 10 pm. Standard bar in H is for E-H and represents 10 pm.

1200

1000
I-
S800
0
w 600

r 400

0
No Control Experimental
Treatment siRNA siRNA

Figure 2.14. Experimental siRNA reduces the number of actin rings on
coverslips by over 95%. RAW 264.7 osteoclast-like cells or osteoclast-like cells
transfected with no siRNA, ineffective siRNA (19941) or effective siRNA (19942)
were fixed after 30 hours and examined for the presence of fluorescent oligo
marker of transfection. The actin rings of the cells with the marker of transfection
present were counted to quantify changes in the number of actin rings formed.
There was a significant decrease in the number of actin rings after treatment with
effective siRNA. Error bars represent standard error. p < 0.05 by student's t-
test.

Figure 2.15. Dendritic Nucleation Model. Upon activation of WASP/Scar family
proteins, the Arp2/3 complex is activated, resulting in actin polymerization and
side-branching of new filaments on existing filaments. As the filaments elongate,
they push the membrane forward. Profilactin is required for filament elongation
at the barbed ends and may be localized to this region by VASP. (ATP-actin -
white; ADP-P-actin orange; ADP-actin -red; profilin black) (Blanchoin L. et al.
Nature. 2000;404:1007-1011) (37)

Table 2.1. PCR Primers Used for Identification of Arp3 Isoforms. The sequences
of primers used for PCR as well as their positions numbered relative to the AUG
start site and the expected product size. All primers were designed against
marine sequences.

such as podosomes (151, 185). VASP can bind directly to G-actin and F-actin as

well as recruit profilactin complexes to the site of actin polymerization. In

addition, VASP is known to enhance Arp 2/3 activity and prevent capping

proteins. VASP is phosphorylated in response to protein kinase A (PKA) and

protein kinase G (PKG) (189, 190). The ability of VASP to be phosphorylated

allows it to be both a positive and negative regulator of actin polymerization.

Calcitonin induces alterations in the cytoskeleton of the osteoclast through the

protein kinase A pathway (191, 192). It is plausible that the disruption of the

actin cytoskeleton by calcitonin could be mediated by VASP. Phosphorylation of

VASP has also been shown to diminish F-actin binding, suppressing actin

nucleation as well as inhibiting Arp2/3 triggered actin polymerization; thus, it can

be a negative regulator of actin polymerization (185). Thus, VASP may play an

important role in the regulation of the translocation of V-ATPase to and from the

plasma membrane.

In summary, the Arp2/3 complex did not bind the same amino acid

sequence of the B1 subunit of V-ATPase as did actin. Further studies are

required to determine if binding exists at another sequence. Two additional

proteins, cortactin and VASP, were identified as having possible key roles in

osteoclast function. Cortactin was found to be preferentially upregulated in

response to RANKL stimulation while VASP was found to associate with the B2

subunit, either directly or indirectly through other V-ATPase subunits or other V-

ATPase bound proteins.

V-ATPase

I
ATP

ADP+Pi
I
__J

'4....+

I~~

catalytic
hexamer

stalk

proton
pathway

Figure 3.1. The structure of V-ATPase. The vacuolar proton ATPase is
composed of 13 or more different proteins and over 20 subunits and consists of
two major functional domains, V1 and Vo. The V1 domain, a peripherally located
cytoplasmic section, contains at least eight different subunits (A-H) and contains
three catalytic sites for ATP hydrolysis. These sites are formed from the A and B
subunits. The Vo domain, a proton channel, is composed of at least 5 subunits
and allows for proton translocation across the ruffled membrane. (Sun-Wada et
al. Biochimica et Biophysica Acta. 2004; 1658: 106-114) (168)

B1 (1-106)
Subunit of
V-ATPase

BI --

Purified
Arp2/3
Complex

IP: Amylose

Figure 3.2. The B1 (1-106) fusion protein of V-ATPase and the Arp 2/3 complex
do not show a direct interaction by binding assay. The B1-MBP fusion protein
and the Arp2/3 complex were incubated together. The sample was then run on
amylose resin to bind the maltose binding protein. The column was then eluted
with maltose. The samples were separated by SDS-PAGE and stained with
Coomasie. The B1 subunit was pulled down in the amylose column but Arp3
was not, indicating a lack of binding between the two proteins.

*- ---

---

IP: MBP

Probe: Arp3

Probe: Arp3

Figure 3.3. The B1 (1-106) fusion protein of V-ATPase and the Arp 2/3 complex
do not show a direct interaction by immunoprecipitation of B1 subunit. The B1-
MBP fusion protein and the Arp2/3 complex were incubated together. The
sample was then incubated with a maltose binding protein antibody. The sample
was then immunoprecipitated with protein G beads which bind the antibody. The
beads were washed and eluted with sodium dodecyl sulfate. The elution was
then probed using the B1 or Arp3 antibodies. B1 was pulled down by the protein
G beads but Arp3 was not, indicating a lack of binding between the two proteins.

Probe: B1

IP: MBP

Probe: B1

- -R

Unstimulated

Cortactin

WASP

N-WASP

VASP

Arp3

GAPDH

Figure 3.4. Cortactin is preferentially upregulated during osteoclastogenesis as
identified by PCR. RAW 264.7 cells were cultured with (stimulated) or without
(unstimulated) RANKL. Cells were harvested and RNA was obtained using
RNAeasy Mini Kit. RT-PCR was performed using primers specific to cortactin,
WASP, N-WASP, VASP and GAPDH (control). Cortactin was the only actin-
associated protein preferentially upregulated in response to osteoclastogenesis.

Stimulated

Figure 3.5. Vasodilator stimulated phosphoprotein is identified to have a possible
interaction with V-ATPase. Signal Transduction Array by Hypromatrix was
probed with biotinylated B2 antibody (work by Sandra Vergara) to identify
possible signal transduction molecules which may interact with V-ATPase.
Vasodilator stimulated phosphoprotein, an actin associated protein, was
identified as having a possible interaction.

B2 Biotin B2 IP: B2 subunit
Streptavidin

VASP

Figure 3.6. Immunoprecipitation experiments with the B subunit of V-ATPase
Suggests a Possible Direct Linkage between VASP and V-ATPase. RANKL
stimulated RAW 264.7 cell lysates were incubated with biotinylated B2 antibody,
pulled down on streptavidin agarose, separated by SDS-PAGE and western
transfer, and probed with the antibodies of various actin related proteins. Of all
the proteins tested, only VASP was pulled down in complex with the B2 subunit
of the V-ATPase.

Table 3.1. PCR Primers Used for Identification of Arp2/3 Related Proteins. The
sequences of primers used for PCR as well as their positions numbered relative
to the AUG start site and the expected product size. All primers were designed
against murine sequences.

to form actin rings and podosomes. Together with the fact that cortactin is

specifically upregulated during osteoclastogenesis (184), these data suggest that

cortactin plays a vital role in osteoclast function.

In summary, we showed that cortactin is required for the formation of the

podosomes and actin rings that are vital for osteoclast function. Cortactin

interacts with Arp2/3 complex and n-WASp as expected in osteoclasts extracts

(186, 188). Novel interactions between cortactin and VASP and cortactin and V-

ATPase were identified. Our data are consistent with cortactin playing a role in

osteoclasts in the integration of cytoskeletal and membrane dynamics.

-WASP

Figure 4.1. Cortactin, N-WASp and Arp2/3 form a synergistic, ternary complex to
initiate actin polymerization. The Arp2/3 complex is inactive in its unbound form.
Activation of the Arp2/3 complex occurs through the N-WASP family of proteins
binding to the Arp2 subunit. Upon activation, a conformation change occurs in
between the Arp2 and Arp3 subunits inducing actin polymerization. Cortactin
binds to the Arp3 subunit and functions to enhance actin polymerization as well
as stabilize the Arp2/3 induced branched actin networks. (Weaver et al. Curr
Biol. 2002; 12:1270-1278) (188)

RANKL
Stimulated

RANKL
Unstimulated

Anti-Cortactin

Figure 4.2. Cortactin is upregulated in response to RANKL stimulation. Cell
lysates were extracted from unstimulated or RANKL stimulated RAW 264.7 cells.
Bradford assay was performed to standardize protein concentrations. Cell
lysates were separated by SDS-PAGE and western transfer and probed with
anti-cortactin antibody. In unstimulated RAW 264.7 cells, cortactin is
undetectable by western analysis; however, upon RANKL stimulation, cortactin
expression is induced.

STUDY OF THE ACTIN-RELA TED PROTEIN 2/3 COMPLEX AND OSTEOCLAST BONE RESORPTION By IRENE RITA MARAGOS HURST A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOIRDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

PAGE 2

COPYRIGHT Copyright 2006 By Irene Rita Maragos Hurst

PAGE 3

iii ACKNOWLEDGEMENTS There are many individuals to whom I owe my success in this scholastic endeavor. First, I would like to thank my parents for their constant support. Through their parenting, I was able to un derstand the importa nce of advanced education, and it is w hat propelled me to further my education. Second, I would like to thank the faculty members that have helped me through this difficult period. My work in research began in embryology under the guidance of Gertrude Hinsch, Ph.D., at the University of South Florida, Tampa, FL. I furthered my research work at the University of Louisville studying microbiological contamination of dental unit air lines under Robert Staat, Ph.D. It was under his guidance that I was able to obtain my MS in oral biology and made the decision to continue my research wo rk and obtain a Ph.D. For the last 5 years, I have had the explicit pleasure working under L. Shannon Holliday, Ph.D., as well as Timothy T. W heeler, DMD, Ph.D., and Caloge ro Dolce, DDS, Ph.D. Their support and guidance have been invaluable The strength of this program, in both clinical and basic science resear ch, has allowed me to learn and utilize many research techniques as well as become an independent thinker. Last, I would like to thank my husband who has trav eled from city to city to support my endeavors. Without him, I would never have made it to this point.

vii LIST OF FIGURES Figure page 1.1 Resorbing os teoclast .................................................................................22 1.2 The OPG/RANK/RANKL triad pla ys an important role in the bone, immune, and vascula r systems..................................................................23 1.3 Binding of the adaptor protein TRAF6 is the initial step in RANKL signaling .....................................................................................................24 1.4 The dynamic nature of the podosomes of ac tin rings .................................25 1.5 In unactivated osteoclasts, V-ATPa se is not present at the plasma membrane but is stored in cytoplasmic vesicles; but upon activation, it is transported via actin filaments to the ruffled membrane............................26 2.1 The Arp2/3 comple x...................................................................................45 2.2 Purification of the Arp2/3 co mplex from human platel ets...........................46 2.3 Upregulation of Arp2 and Arp3 during ost eoclastogenes is........................47 2.4 Two isoforms of Arp3, Arp3 and Ar p3-beta, are present in unactivated and activated os teoclasts...........................................................................48 2.5 Arp2/3 complex is present in actin rings of osteocla sts..............................48 2.6 Arp2/3 complex is enriched relative to F-actin near the sealing zone........49 2.7 Arp2/3 does not co-localize wi th vinculin in actin rings..............................50 2.8 Treatment with chem ical agents, cytochalasin B, echistatin, and wortmannin, causes a disruption of the actin rings of osteoclasts..............51 2.9 The Arp2/3 remains co-localized in the actin based podosomal core regardless of actin ring di sruption by wo rtmanni n......................................52 2.10 Wortmannin and echistatin treatment of osteoclasts results in a decrease in the number of actin rings ........................................................................52

PAGE 8

viii 2.11 siRNA 19942 but not 19944 reduces the Arp2 cont ent of osteoclast-like cell extract 70% after 30 hours compared wit h acti n..................................53 2.12 Actin rings are disrupt ed in Arp2 k nockdown.............................................54 2.13 Actin rings are disrupted in marro w osteoclasts on coverslips or on bone slices by siRNA di rected agains t Arp2.......................................................55 2.14 Experimental siRNA reduces the number of actin rings on coverslips by over 95%....................................................................................................56 2.15 Dendritic nu cleation model .........................................................................57 3.1 The structure of V-AT Pase.........................................................................70 3.2 The B1 (1-106) fusion protein of V-ATPase and t he Arp2/3 complex do not show a direct intera ction by bindi ng assay...........................................71 3.3 The B1 (1-106) fusion protein of V-ATPase and t he Arp2/3 complex do not show a direct interaction by immunoprecipitation of B1 subunit...........72 3.4 Cortactin is preferentially upregu lated during osteoclastogenesis as identified by PCR.......................................................................................73 3.5 Vasodilator stimulat ed phosphoprotein is identified to have a possible interaction wit h V-ATPa se..........................................................................74 3.6 Immunoprecipitation experiments with the B subunit of V-ATPase suggest a possible direct link age between VASP and V-ATPase..............75 4.1 Cortactin, N-WASP, and Arp2/3 form a synergistic, ternary complex to initiate actin pol ymerizat ion........................................................................88 4.2 Cortactin is upregulated in re sponse to RANKL stimulat ion.......................89 4.3 Cortactin co-localizes with the podosomal core proteins, actin and the Arp2/3 comp lex..........................................................................................90 4.4 siRNA 120649, but not a control siRNA (120653), effectively knocks down the cortactin content to an undetec table level of osteoclast-like cell extract after 30 hours co mpared with actin................................................91 4.5 Actin rings are disrupted in cortactin knockdow n.......................................92 4.6 An siRNA known to downregulate co rtactin (Ambion) effectively knocks down the cortactin content of osteoclast-like cell extract to an undetectable level after 30 hour s compared wit h actin..............................93

PAGE 9

ix 4.7 Actin rings are disrupt ed in cortactin knockdow n.......................................94 4.8 Transformation and purification of GST-cortactin fu sion prot ein................95 4.9 Immunoprecipitation experiments with GST-cortactin show a linkage between cortactin and Arp3, VASP, NWASP, and the E subunit of VATPase ......................................................................................................96 5.1 The ENA/V ASP family .............................................................................108 5.2 VASP is present in the acti n rings of ost eoclasts .....................................109 5.3 VASP is phosphorylated at Seri ne 157 in response to calcitonin treatment and results in the disr uption of the ac tin ring ............................110 5.4 Calcitonin induces a three fold increase in phosphorylation levels of VASP at Seri ne 157................................................................................. 111 5.5 Identification of VASP null mice fr om breeding of homozygous females with a heterozy gous male ........................................................................112 5.6 Osteoclasts of mice lacking the VASP gene are able to form actin rings and respond to calcitonin in the sa me fashion as c ontrol ce lls.................113 5.7 Evl, a member of t he ENA/VASP family, is up regulated in response to osteoclasto genesis ..................................................................................114

PAGE 10

x Abstract of Dissertation Pr esented to the Graduate School Of the University of Florida in Partial Fulfillment of the Requirements for t he Degree of Doctor of Philosophy STUDY OF THE ACTIN-RELATE D PROTEIN 2/3 COMPLEX AND OSTEOCLAST BONE RESORPTION By Irene Rita Maragos Hurst August 2006 Chair: Lexie Shannon Holliday Major Department: Medi cal Sciences--Molecular Cell Biology To resorb bone, osteoclasts form an extracellular acidic compartment segregated by a sealing zone. This is dependent on an actin ring that is composed of filamentous actin organi zed into dynamic structures called podosomes. The molecular basis of ac tin rings and their association with vacuolar H+-ATPase (V-ATPase) mediat ed-acidification during bone resorption were examined. Immunoblotting and immunocytochemic al studies showed for the first time that the actin-related protein (A rp) 2/3 complex is upregulated during osteoclastogenesis and expressed in ac tin rings. Knockdowns of Arp2, a component of the Arp2/3 complex, with s hort interfering RNAs (siRNAs) revealed that it is essential for actin ring formati on. No direct associations between VATPase and Arp2/3 complex were detected. Two proteins involved in regulating Arp2/3 mediated actin polymerization were iden tified in in vitro binding studies as

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xi interacting with V-ATPase: cortactin and vasodilator-stimulated phosphoprotein (VASP). Cortactin binds and activates Arp2/3 complex. It was upregulated during osteoclastogenesis and localized to the cores of podosomes. siRNA knockdowns showed that it was required fo r actin ring formation. Binding studies suggest that it may interact with V-AT Pase indirectly through the glycolytic enzyme aldolase. VASP was shown to be present in actin rings and phosphorylated in response to calcitoni n, which disrupts actin rings. VASP knockout mice did not demonstrate ost eoclast or bone defects. ENA-VASP-like protein (Evl), a protein closely rela ted to VASP, was al so expressed in osteoclasts and may substitute for t he lack of VASP. These data demonstrate that the Arp2/3 complex and cortactin play significant roles in osteoclastic bone resorption and may provide targets for therapeutic agents des igned to limit the activity of osteoclasts.

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1 CHAPTER 1 INTRODUCTION Bone remodeling is a result of the processes of bone resorption and formation and primarily involves two types of cells (1). Osteoblasts, cells of the mesenchymal lineage, form bone and r egulate osteoclast differentiation and activation (1). Osteoclasts, the bon e resorbing cells, are derived from hematopoietic precursors and are close relatives of macrophages (2-4). Upon activation, the osteoclast under goes profound reorganizations (5, 6) and becomes polarized, forming morpholog ically and functionally distinct basolateral and resorptive domains (3, 7, 8) (Figure 1.1). The bone-apposing resorptive domain contains three primar y functional structur es: the sealing or clear zone, the ruffled membrane, and integrin-mediated adhesions. The ability of the osteoclast to resorb bone is dependent on it s ability to generate an extracellular acidic com partment between itself and the bone (7-9). This local acidification is maintained by t he presence of the sealing zone, which forms tight contact with the bone surface (6, 9, 10). The acidic pH of this compartment is created by vacuolar H+ATPases (V-ATPases ) (8, 11), in the ruffled membranes which are bounded by sealing zones. V-ATPases pump protons into the extracellular space, solubilizing hydroxya patite crystals (2), and providing the acidic environm ent required for the acid cysteine

PAGE 13

2 protease, Cathepsin K, whic h is involved in the dige stion of the organic bone matrix (2, 5, 9, 12). Osteoclast Differentia tion: RANKL Signalling Osteoclasts differentiate from ci rculating hematopoietic stem cells that are recruited to the bone to fuse and form mu ltinucleated osteoclasts (13-16). The osteoclast has phenotypic feat ures that distinguish it from other members of the macrophage/monocyte family such as the ex pression of tartrate-resistant acid phosphatase (TRAP) and the calcitonin recept or and the formation of the ruffled membrane (4, 13). Osteoclastogenesis is dependent on two important fa ctors, receptor activator of nuclear factor kappa B lig and (RANKL), a tumor necrosis factor (TNF) related cytokine, and co lony stimulating factor-1 (CSF-1) (Figure 1.2) (4, 13, 17). These factors induce the osteoc last to express genes specific for osteoclastogenesis such as thos e encoding cathepsin K, TRAP, and 3-integrin (3). Once osteoclastogenesis has occurr ed, RANKL and interleukin 1 function to increase the osteoclast survival time by inducing nuclear factor kappa B (NF B) activity (13). RANKL is a TNF-related type II transmembrane protein found on osteoblasts either as a su rface protein or in a prot eolytically released soluble form (1, 4, 13). The expression of RANKL coordinates bone remodeling by stimulating bone resorption in the osteoclast by bindi ng and activating the tumor necrosis factor receptor (TNFR)-rela ted protein, RANK, a transmembrane receptor expressed on the surface of hem atopoietic precursors (1, 4, 13). The

PAGE 14

3 requirement of these two proteins in os teoclastogenesis is indicated as mice deficient in either RANK or RANKL are severely osteopetrotic with the inability to resorb bone (1). In addition, anti bodies neutralizing RANKL inhibit bone resorption induced by stimulants such as parathyroid hormone (PTH) and prostaglandin E2 (PGE2) (16). Activation of RANK leads directly to the expression of os teoclast specific genes by the association of various TNF -receptor associated factor proteins (TRAFs) relaying the signal to at least fi ve major signaling cascades: inhibitor of NFB kinase (IKK), c-Jun N-terminal kinase (JNK), p38, extracellular signalrelated kinase (ERK), and Src pathways (1, 13, 17) (Figure 1.3). The initial step is the binding of TRAFs, cytoplasmic adaptor proteins, to specific domains of the cytoplasmic portion of RANK, in which three putative TRAF binding domains have been identified (1, 13, 17). The binding sites of TRAF-2, -5, and -6 to RANK have been identified; however, only muta tions in TRAF-6 result in a loss of osteoclast activity, resulting in osteopet rosis (1, 13, 18). TRAF6 is the key adaptor linking the expression of NFB and activator protein-1 (AP-1) to RANK (1, 13). Osteoclastogenesis is inhibite d by mutations in the p50/p52 component of NFB or the c-Fos component of AP-1, resulting in osteopetrosis (1, 13). TRAF2 and 5 are also able to activate NFB pathways, but thei r contributions to osteoclastogenesis are minor TRAF3, however, has been shown to serve as a negative regulator of NFB activation (1, 13). Activation of the protein kinase p38 by RANK results in the activation of the transcriptional regulator mi/Mitf (13). This regul ator controls the gene

PAGE 15

4 expression of both TRAP and Cathepsin K, which are both required for the osteoclast phenotype (13). ERK-1 kinase is also regulated by RANKL through upstream activation by MEK-1 (13). ER K appears to negatively regulate osteoclastogenesis as inhibitors of ERK potentiate RANKL induced osteoclastogenesis (13). Src protein binds TRAF6, pe rmitting RANK-mediated signaling to continue through the tyrosine phosphorylation of phos phatidylinositol 3-OH kinase (PI3K) and the serine/threonine protein kinase (A KT) (13). Both PI3K and AKT are involved in various cellular processe s, such as motility, cytoskeletal rearragements, and cell survival (13). Mu tations in the Src protein have been shown to cause osteopetrosis in mice ( 13, 19). Multiple factors are res ponsible for both positively and negatively regulating osteoclastogenesis by affecting expression of RANKL. Interluekin-1, c-Fms, tumor necrosis factor (TNF), prostaglandin (PG) E2, and transforming growth factor (TGF)activate surface receptors on the osteoclast to potentiate osteoclastogenesis and bone re sorption (13, 17). It is known that IL1-R and TNFR1 signal through the TRAF6 pathw ay and have a synergistic effect on RANK mediated TRAF6 activation, while c-Fms and TGFaffect osteoclastogenesis by upregul ation of its components, such as the surface receptor RANK (13, 17). Negative regulation of ost eoclastogenesis through RANKL occurs by osteoprotegerin (OPG), a solu ble protein secreted by osteoblasts (1, 13, 17). OPG is a TNFR-related protein and acts as a decoy receptor by binding to

PAGE 16

5 RANKL and blocking its ability to bind RA NK (1, 13, 17). It is controlled hormonally by bone anabolic agents such as bone morphogenic proteins (BMPs) (13). These factors caus e an overproduction of OPG which blocks osteoclast differentiation, leading to osteopetrosis (13). Hormonal Control of Bone Resorption Stimulation of osteoclastogenesis by calciotropic factors and proresorptive cytokines such as parathyroid hormone related peptide (PTHrP), parathyroid hormone (PTH), interleukin (IL)1b, tumor necrosis factor (TNF), 1,25 dihydroxyvitamin D3, or prostaglandin (PG) E2 (13, 20, 21), acts indirectly via osteoblastic stromal cells (16, 22) by i nducing mRNA expression of RANKL. In converse, factors such as estrogens c ause a decrease in RANKL expression and an increase in OPG expression, causing reduced numbers of osteoclasts (13). The cytokine, thrombopoietin, has al so been identified to induce OPG expression. Calcitonin also is known to inhibit bone resorption (13). Mechanism of Action of Osteoclast Once the osteoclast attaches to bone, there is segregation of an extracellular compartment between it and th e bony surface (1). The area of tight adhesion segregating this extr acellular compartment is termed the sealing zone (1). Bounded by the sealing zone is t he ruffled membrane (1). The ruffled membrane is a convoluted membrane pa cked with vacuolar proton ATPase (VATPase), the osteoclast proton pump ( 23). The protons, which are pumped by the V-ATPase and are responsible for bone demineralization, are obtained by various mechanisms. One mechanism is the hydration of carbon dioxide to

PAGE 17

6 carbonic acid by carbonic anhydrase II (C A II) (3). The carbonic acid then dissociates into protons and bicarbonate ions. Although traditionally described as the primary mechanism of proton prod uction in the osteoclast, osteopetrosis caused by mutations in carbonic anhydras e II is mild and improves with age (24, 25). This would suggest an alternative source of protons is available. Osteoclastic glycolysis provides the me chanism for an alternative source of protons. In the glycolytic process, one or two hydrogen ions are generated for every ATP molecule produced or glucose mo lecule consumed respectively (26). Recent data indicate that several glyco lytic enzymes bind directly to the VATPase and that V-ATPase assembly requires the glycolytic enzyme aldolase (26, 27). These data s uggest that V-ATPase, by directly interacting with glycolytic enzymes, forms an acidifying me tabolon. Regardless of their source, at the resorptive membrane, the protons are utilized by the V-ATPase to acidify the extracellular compar tment (23, 28). At the basolateral membrane, bicarbonate is exchanged for chloride ions in an energy dependent manner (3). The chloride ions, which have entered the osteoclast, pass into the extracellular compartment through a volt age gated anion channe l coupled to the V-ATPase (3, 23). The V-ATPase generates a membr ane potential and the chloride channel dissipates this potential formed by the pr otons from the V-ATPase allowing the pH to decrease in the extracellular com partment to approximat ely 4.5 (3, 23). The highly acidic nature of the extrac ellular compartment dissolves the bone mineral, which in turn, exposes the organic matrix of the bone (3). Cathepsin K, an acid cysteine proteinase generated by the osteoclast, is then able to degrade

PAGE 18

7 the bone matrix, which is primarily composed of type I collagen and non collagenous proteins (3). The degraded bon e, both protein and mineral, are then transcytosed through the osteoclast and secreted into the microenvironment through the basolateral membrane (3). Sealing Zone The sealing zone segregates the acidic resorption compartment from the surrounding environment, analogous to creati on of an extracellular lysosome (7, 8). By electron microscopy, this ar ea of the plasma membrane demonstrates extremely tight adhesion, less than 10 nm, between the plasma membrane and the adjacent bone surface (29). The mo lecular mechanisms accounting for the sealing zone are still unknown. Several actin binding proteins including vinculin and gelsolin, have been localized to the sea ling zone (30). In addition, there is much evidence that the formation of an ac tin ring is required for formation of the sealing zone (5-7, 31, 32). When acti n rings are disrupted by calcitonin, herbimycin A, or bisphosphonates, ruffled membrane formation and bone resorption are inhibited (31). Thus, this region is critical in osteoclastic bone resorption. Podosomes Podosomes are small, discrete but highly dynamic F-actin based structures. Structural studies indicate that there are two main domains of podosomes, a cylindrical dense actin core with a surrounding ring enriched with v3 and focal adhesion proteins, such as int egrins, vinculin, paxillin, and talin (33, 34). Along with actin, additiona l core components in clude Wiskott-Aldrich

PAGE 19

8 Syndrome protein (WASP) family members, the Actin Related Protein (Arp) 2/3 complex, and cortactin (35, 36). The core and ring may be linked by a bridging protein such as -actinin. Peripheral to the ring domain, it is hypothesized that a cloud of monomeric actin resides (33, 37). Although podosomes are typically found in monocytic cells and are not specific to the osteoclast (38), it is only in the osteoclast that they arrange themselves into a defined actin ring and are associated with a sealing zone (33, 39). Podosom es can also be found or induced in several other cell types, such as endothelial cells, and cells transformed with v-src (33, 35, 40, 41). Podosomes are relatively sma ll with a diameter of 0.5-1 m and a depth of approximately 0.2-0.4 m (33). Although small, they are found in great numbers in osteoclasts (33, 42). Current res earch suggests that the actin ring of osteoclasts is formed by a rearrangement and fusion of individual podosomes with a slightly different 3-dimensional struct ure (43). This structure still maintains an actin based core but the cloud of pr oteins is now propos ed to surround the entire actin ring structure rather t han each individual podosome (43). These actin ring structures can become as large as 4 m in height and diameter (43). Regardless, podosomes are highly dynamic turning over every 2-12 minutes, with the the length of the actin core turning over mult iple times within the lifespan of the podosome, likely facili tated by gelsolin (44) and dynamin (36, 42). Figure 1.4 depicts the dynamic nature of podosoma l structures in the actin ring. Rhodamine actin was introduced into saponin-permeabilized activated osteoclasts to allow for the fluorescent visu alization of incorpor ation of actin into

PAGE 20

9 the actin ring. If the actin ring is stat ic, no incorporation would occur; however, within 10 minutes, the rhodamine actin wa s incorporated into the actin ring, verifying the dynamic nature of the actin ri ng. To confirm this dynamic nature, the activated osteoclasts were treated with latrunculin A, which binds monomeric actin (45, 46). Due to the inability to add new actin monomers, a loss of podosomal structures and actin ring is observed. Podosome assembly and disassembly occurs from front to end wit h F-actin continuously adding at the leading edge and treadmilling through to the basolateral region (33, 35). It is of note that podosomes are only present on adherent cells, indicating that attachment may be the initiating fact or with regulation occurring by a variety of mechanisms. Signalin g pathways which regulate po dosomal formation include Rho family GTPases, such as R hoA, Rac1, or CDC42, and tyrosine phosphorylation by Src or Csk. (35, 47). It has been noted that both dominant active and inactive mutations in Rho fa mily GTPases affect the formation and localization of podosomes; however, t he mechanism of disruption has been shown to be dependent on cell type (47). In addition, the use of Src kinase inhibitors causes failure of podosom es while the use of phosphotyrosine phosphatase inhibitors induces podosomal formation (48, 49). Transportation of V-ATPase to the Ruffled Membrane The vacuolar H+-ATPase plays a vital role in bone resorption, as it is the proton pump responsible for acidification of the extracellular compartment and demineralization of the bone (8, 9, 11, 12, 23). In unactivated osteoclasts, VATPase is not present at the plasma me mbrane but rather stored in cytoplasmic

PAGE 21

10 vesicles (23, 50). In the inactivated stat e, the V-ATPase is bound to F-actin (9, 51); but upon activation, the mechanism by which translocation of actin and VATPase to the plasma membrane occurs is still unknown (Figure 1.5) (9). Ruffled Membrane The ruffled membrane is the resorption or ganelle of the osteoclast (8). Its name is derived from the brush border-like appearance of the plasma membrane (8). The ruffled border is formed by the fu sion of intracellular acidic vesicles with the plasma membrane, adjacent to the bone surface (6, 8, 11). The fusion of these vesicles causes an enr ichment of vacuolar proton ATPase in the plasma membrane (7, 11), which pumps protons to acidify the resorption compartment (23, 50). Osteoclast Adhesion Adhesion of the osteoclast to bone is integral in the resorption process. The integrin, v3, is a key player in adhesion of the osteoclast to bone (30, 52) by recognizing Arginine-Glycine-Aspartic Ac id (RGD) moieties in extracellular matrix (ECM) proteins (53). This int egrin has been localized to the basolateral membrane, intracellular vesicles and ruffl ed border (30, 54). Bone resorption, osteoclast formation and attachment have been shown to be inhibited by disintegrins, blocking antibodies, and RGD mimetic peptides, indicating the importance of v3 in osteoclast adhesion ( 55-57). Echistatin, an RGD containing disintegrin which binds v3 tightly, induces osteoclastic detachment from its substrate (55, 58) The use of echistatin in vivo causes an inhibition of bone resorption without significantly alteri ng the number of osteoclasts (59),

PAGE 22

11 resulting in a decreased osteoclastic efficiency without effects on osteoclast differentiation and recruitment (60). In addition, a deletion of the 3 integrin subunit did not affect osteoclast recruitm ent, which is thought to be mediated by 5, or the formation of reso rption lacunae (52). The 3 -/mice did show decreased bone resorption, abnormal ruffled membranes, and increased osteoclast number, most likely caused from stimulation by hyperparathyroidism secondary to the hypocalcemia produced by decreased bone resorption (52). Skeletal remodeling in the 3 -/mice proceeds even in the absence of v3; it is hypothesized that an adequate resorption rate is achieved by the increased number of osteoclasts, even in the presence of decreased resorption per osteoclast (52). Howe ver, with age, the compensation decreases, and osteosclerosis occurs (52). Although onc e thought to mediate the extremely tight seal of the sealing zone, Lakkakorpi et al. (57) and Masarachia et al. (59) have shown the specific exclusion of v3 from the sealing zone. However, its absence from the sealing zone does not preclude its ability to cause a visible disruption of the sealing z one as was shown by Nakamura et al. (60). It seems likely that proper stimulat ion of integrin-based sig nal transduction pathways normally plays a role in the acquisition and maintenance of osteoclast polarity during bone resorption (61). Osteoclasts and Disease As previously stated, bone homeos tasis is dependent on a delicate balance between bone resorption and bone formation (62). When one is in excess of the other, bone diseases occur. Most commonly, skeletal diseases are

PAGE 23

12 a result of an excessive am ount of bone resorption, result ing in osteoporosis (1, 62). Osteoclastic bone diseases are c aused by reduced number of osteoclasts, reduced or loss of function or overac tivity of osteoclasts (62). There are several diseases which result in reduced osteoclast activity and thus, osteopetrosis, which often leads to brittle bones and fractures (62-64). Autosomal recessive malignant osteopetrosis is a result of a mutation in the TCIRG1 gene (65, 66). This gene enc odes for the 116 kD a3 subunit of VATPase (65, 66). The resultant phenoty pe is osteoclast-rich but with poor resorptive abilities (65, 66). Autoso mal dominant osteopetrosis type II (AlbersSchonberg disease) results from a muta tion in the CLCN7 gene, which encodes for the CLC7 chloride chann el (67-69). As a result of this mutation, normal numbers of osteoclasts are present; how ever, resorption is inhibited as acidification of the resorption lacunae is hindered (67-69). Autosomal dominant osteopetrosis type I has been linked to a gain of function mutation in the LRP5 gene (70-72). In this disease, osteoc last function is not impaired; however, abnormally low numbers are present (70). It is hypothesized that the mutations in the LRP5 gene alter the osteoblast, decreasing the potential to support osteoclastogenesis (70). Pycnodyostosis has been showed to be a result from mutations in the Cathepsin K gene, an ac id cysteine protease responsible for the degradation of organic bone matrix (73-75). A deficiency in this protease results in elevated numbers of os teoclasts and disorganized bone structure (73-75). Another osteopetrotic disease is t he autosomal recessive syndrome of osteopetrosis with renal tubular acidosis and cerebral calcification; which is, most

PAGE 24

13 frequently referred to as carbonic anhydrase isoenzyme II deficien cy (24, 25, 76). CAII is responsible for one of the mechani sms by which the protons, which are responsible for acidification of the re sorption compartment, are produced (3). Thus, prevention of normal acidification occu rs (24, 25, 76). A decrease in osteoclast number may result from defects in the CSF1 (colony stimulating factor) gene (62). De fect in this gene in the murine model results in a broad spectrum of pathology fr om a delay in osteoclast formation to a complete inhibition of osteoclast forma tion. In addition, polarization can be affected and there may be a loss of the ru ffled border (62). However, to date, there have been no human case s of osteopetrosis attri buted to a lack of CSF-1 (62). The diseases of increased osteoclast ic activity include Pagets disease (PD), expansile skeletal hyper-phosphatasia and famili al expansile osteolysis (FEO) (77). The second most common bone disease, after osteoporosis, is Pagets disease of bone (77). This diseas e primarily occurs as a result from a mutation in SQSTM1, which encodes sequestosome 1, an ubiquitin binding protein involved in multiple signaling pat hways, including RANKL, IL-1 and TNF (78, 79). However, recent cases hav e reported a mutation in the TNFRSF11A gene as well which encodes RANK (80-83) Unlike Pagets, familial expansile osteolysis and expansile skeletal hyperphosphatasia result primarily from defects in the TNFRSF11 A, which is the gene encodi ng RANK (80, 83). Regardless of mutation location, these defec ts result primarily in an enlargement of the osteoclasts with an increased number of nuclei ( 80-83). In addition, there

PAGE 25

14 can be an increase in osteoclast number as well as in activity (80-83). A striking finding in both FEO and PD are nuclear inclusions similar to those seen by viral infections (83). The osteoclast is also im plicated in diseases in which skeletal pathology results from inflammation (84-86). In rheumatoid diseases, such as rheumatoid arthritis, seronegative spondyloarthropat hies, and systemic lupus erythematosis, as well as periodontal disease, the osteoclast has been identified as the dominant cell type which mediates th e inflammatory bone loss (84-86). Activation of the osteoclasts occurs due to increases in proinflammatory cytokines, such as TNF, Interferon (INF), and interleukins, which then modulate expression of RANKL and OPG (84, 85). Treatment of Osteoporos is and Osteopetrosis Osteoporosis occurs as a result of an imbalance in the bone remodeling cycle resulting in excessive bone loss (87-89). For the past decade, the treatment of osteoporosis wa s based on the retardation of bone mineral density loss (88). However, bone formative medi cations have recently come on the market. The anti-resorptive medications slow bone resorption and formation, but the effect on formation is less dramatic, allowing bone formation to exceed bone resorption and bone density to increase modestly (88). Anti-resorptive medications include the bi sphosphonates, estrogens, sele ctive estrogen receptor modulators, and calcitonin. Calcium is important in the preventi on and treatment of osteoporosis (90, 91). Adequate calcium is important for i ndividuals at all ages Individuals, with

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15 high calcium intake as children, hav e increased bone mass, which is an important variable in future fracture risk, as the risk for osteoporotic fractures is inversely related to bone mineral densit y (91). Post-menopausal use of calcium has been shown to decrease bone loss and prev ent tooth loss but there is little or no reduction in the risk of spinal fractu res (90, 91). Calcium intake should be between 1000-2000 mg/daily (91) Although calcium may slow the loss of bone mineral density, most physicians suppor t the use of additional pharmacologic intervention to prevent/treat osteoporosis (91). Estrogens and SERMS function as estrogen receptor agonists (88, 89, 92, 93). Estrogen therapy, also known as hormone replacement therapy, has been approved primarily for the prevention of os teoporosis. It has also been shown to increase bone density modestly, reduc e bone loss and reduce the risk of fractures in postmenopausal women (92, 93). Selective Estrogen Receptor Modulators (SERMS) bind to the estrogen receptor. Although their mechanism of action is not fully understood, these agents may f unction by inducing conformational changes in the estrogen re ceptor, causing differential expression of specific estrogen-regulated genes in different tissues (92, 93). SERMS (raloxifene) are used for both the pr evention and treatment of post-menopausal osteoporosis. They function like the es trogens but without the disadvantages of estrogens, such as the increase in uterine cancer (92, 93). Raloxifene has been shown to increase bone mass and reduce spinal fractures; however, as of yet, there is no evidence indicating a decreas e in non-spinal fractures (92, 93). Recent data have shown signific ant risks for breast cancer, venous

PAGE 27

16 thromboembolism and stroke with the us e of estrogens and SERMS (94, 95). Data on the incidence of breast cancer hav e identified an increa sed risk in ductal and lobular cancer with the use of medium potency estrogens and an increase in lobular cancer with low potency estrogens (94). In addition, if additional risk factors are added, such as alcohol consumption and th e use of oral contraceptives, an increase in all three brea st cancer subtypes (ductal, lobular or tubular) was observed (94). An examination of the lite rature identified increased risks of thromboembolism in pati ents in their first year of therapy and those taking an estrogen-progesterone or high dose estrogen preparation (95). Route of administration also increases the risk as oral administratio n had significantly higher incidence of thromboem bolism than transdermal (95). The bisphosphonates, alendronate, i bandronate and risedronate, are used for the prevention and treatment of postmenopausal bone loss (88, 89, 92, 93, 96, 97). They function to slow bone loss, increase bone density and reduce the risk of skeletal fractures (97). There ar e two main categories of bisphosphonates (96). Amino bisphosphonates inhi bit osteoclastogenesis by blocking isoprenylation of Rho and Rap and indu cing apoptosis wh ile the non-amino bisphosphonates are metabolized to cytot oxic ATP analogues thus inducing cell death (69, 98). Although very effective in the treatment of osteoporosis, the use of bisphosphonates carries significant side effects (99). Several studies have demonstrated a high risk of gastric duodenal, and esophageal ulcers with administration (100). In addition, two percent of bisphosphonate users demonstrate acute systemic inflammatory reactions, ocular complications, acute

PAGE 28

17 and chronic renal failure, and electrolyte imbalances (99). Osteonecrosis of the mandible or maxilla has recently been iden tified as sequelae of treatment with bisphosphonates (99, 101-103). Thes e lesions presented as non-healing, usually as the result of dental surgical intervention (99, 101, 102). Although the large majority of these pat ients were receiving parenter al administration of the drug, several patients were on oral dos es (99, 101, 102). Many researchers strongly support further studies to ident ify the risks and benefits of continuing bisphosphonate therapy (99, 101-103). Calcitonin is also used for the prev ention and treatment of osteoporosis (104, 105). This naturally occurring hor mone is involved in calcium regulation and bone metabolism (104, 106, 107). It is administer ed nasally rather than orally, as it is a protein and would be degraded prior to its function (104). Calcitonin has been shown to increase bon e mass and reduce spinal fractures. In addition, studies have shown a decrease in pain post-fractur e with the use of calcitonin (105). Non-spinal fracture s, however, have not been shown to be reduced with calcitonin treat ment (105). A resistance to continuous treatment with calcitonin, with a loss of inhibitory effects on bone resorption, has been shown to occur within 12-18 months afte r initiation of treatment due to a downregulation of the calcitonin receptor, by both internalization of the receptor and a reduced concentration of de novo rec eptor synthesis (106, 107). Recent data have shown that this resistance can be avoided by the use of intermittent administration of calcitonin, as calcitonin receptor mRNA expression returns to normal by 96 hours after discont inuation (106, 107).

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18 Teraparatide (Forteo), par athyroid hormone [1-34], is a newly approved medication to treat osteoporosis via bo ne formation (108-110). Its mechanism of action is to increase bone formation by t he osteoblasts (108-110). It has been shown to stimulate bone formation and in crease bone mass to a greater extent than the anti-resorptive agents (108-110). Reductions in spinal and non-spinal fractures have been shown (108-110). Like calcitonin, it is a peptide but it is given by injection daily whic h is a disadvantage of this treatment (108, 110). The most common adverse effects of treat ment with teraparat ide include headache, nausea, dizziness, and cram ping; however, only dizziness and cramping differed from placebo in a randomized clinical trial (111). Other less common complications include hypercalcemia and hyperuricemia (111). These complications can often be inhibited by a reduction of the dosage but may require complete cessation of the drug (111). An imal studies have shown an increased risk for osteosarcoma with the use of te raparatide; however, osteosarcoma has not been identified in over 2800 patients in human clinical trials (111). Several new treatment modalities are on the horizon for osteoporosis. Zolendronic acid, an injectable bisphosphona te, is currently being studied. It has been shown to increase bone mineral density modestly as do the other bisphosphonates (93). In addition, strontium ranelate, the only current drug known to decrease bone resorption and increase bone formation concomitantly, has just recently finished Phase III trials (93, 112). It has been shown to reduce both vertebral and non-vertebral fractures (93). Its efficacy and safety have been shown; and therefore, it should be marketed soon (112). In addition, as the proof

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19 of concept for bone anabolic therapy has been established with the use of parathyroid hormone, ot her parathyroid hormone analogues are being investigated as well as the development of non-peptide small molecules targeted against the parathyroid hormone receptor. The treatment of osteopetrosis has fo cused on the stimulation of host osteoclasts with calcium restriction, calc itrol, steroids, parathyroid hormone, and interferon (113, 114). Infantile maligna nt osteopetrosis has also been treated with bone marrow transplantat ion (113, 114). Coccia et al. (115) documented a case of successful bone-marrow transplantati on in a five month old girl in 1980. Prior to transplantation, the patient exhibited anemia, thrombocytopenia, low serum calcium and elevated serum alkali ne phosphatase and acid phosphatase all of which normalized within 12 weeks po st-transplantation (115). In addition, histologic sections prior to transplantat ion showed an increa se in osteoclast number but no bone resorpti on occurring (115). Post -transplantation, active osteoclastic bone resorption occurred (115) Unfortunately, although there have been some reports of successful treatment of osteopetrosis, most research indicates ineffectiveness of treatment and patients are us ually given poor prognosis (113). Difficulty in treatment also stems from the mult iple etiologies of osteopetrosis, and therefore, treatment must be individualized to each patient (113). Osteoclasts and Dentistry Osteoclasts play a significant role in the oral cavity, both through physiologic and pathologic proce sses. The osteoclast is central to the bone loss

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20 observed in periodontal disease. In the inflammatory process in the periodontium, recent data have show n increased levels of RANKL and decreased levels of OPG in patients wit h periodontal disease (116-118). Recent data have also identified RANKL expressi on on both T and B lymphocytes (117). It is suggested that the bacterial biofil m initiates an immune response with expression of RANKL which in turn st imulates osteoclastogenesis and bone resorption (117). This hypothesis is c onfirmed by data show ing an abrogation of bone resorption when RANKL is inhi bited or knocked out (117). Dental root resorption is anot her pathologic process mediated by the osteoclast. Dental root resorption is fair ly unpredictable and t he etiology is still unknown (119). Recent studies however identify increased le vels associated with the IL-1 gene (120). Studies on RANKL and OPG expression when heavy forces are applied during or thodontic tooth movement s how increased levels of RANKL to OPG associated with root resorption (121). In contrast, root resorption has been shown to be inhibited with echist atin treatment, a known inhibitor of osteoclasts (119). Osteoclasts do not always play a pathologic role in the oral cavity. In fact, resorption can be accelerated or inhibite d based on the needs of the orthodontic patient. Several studies have shown that osteoclastic bone resorption can be decreased with the addition of chemical mediators or cyt okines (122-126). Mice lacking the TNF type 2 receptor show less bone resorption than wild type mice (126). Addition of OPG to the periodontal tissues of mice has also been shown to decrease osteoclastogenesis (125). In a ddition, inhibition of orthodontic tooth

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21 movement has been observed when tr eated with matrix metalloproteinase inhibitors, echistatin or an RGD peptide (123). In contrast, orthodontic tooth movement can be accelerated by the remo val of OPG. Compared to wild type OPG littermates, OPG knock out mice sh ow increased osteoclast number and increased alveolar bone resorption (127). In the future, the power of the osteoclast may be able to be harnessed to enhance the treatment of the dental patient. General Purpose of Research The general purpose of the work pres ented in this dissertation has been to learn more about the actin ri ng of osteoclasts, its char acteristics and composition and requirements for formation. In addition, we sought to identify a relationship between components of the actin ring and V-ATPase, another specialized structure of the osteoclast.

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22 Figure 1.1. Resorbing osteoclast. Once the osteoclast attaches to bone, there is segregation of an extracellu lar compartment between it and the bony surface. The area of tight adhesion segregating this extracellular compartment is termed the sealing zone. Bounded by the seali ng zone is the ruffled membrane. The ruffled membrane is a convoluted memb rane packed with vacuolar proton ATPase (VATPase), the osteoclast proton pump (3). Bone degradation is initiated by hydration of carbon dioxide to carbonic acid by carbonic anhydrase II (CA II). The carbonic acid then dissociates into protons and bicarbonate ions. At the apical membrane, the pr otons are pumped into the extracellular compartment via the V-ATPase. At the basolateral membrane, bicarbonate is exchanged for chloride ions in an energy dependent manner The chloride ions, which have entered the osteoclast, pass into the extr acellular compartment through an anion channel coupled to the V-ATPase. The protons and chloride ions form hydrochloric acid and reduce the pH in the extracellular compartment to approximately 4.5, which a llows the deminera lization of the bone mineral and exposes the organic matrix of the bone Cathepsin K, an acid cysteine proteinase, is then able to degrade the bone matrix. The degraded products, collagen and calcium, are then transcytosed through the osteoclast and secreted into the microenvironment through the basol ateral membrane. (Teitelbaum et al. J Bone Miner Res 2000; 18:344-349) (3)

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23 Figure 1.2. The OPG/RANK/RANKL triad pl ays an important role in the bone, immune, and vascular systems. In t he bone system, the interaction between OPG and RANKL promotes eit her osteoclast differentiation and survival or osteoclast apoptosis. (Theoley re et al. Cytokine and Growth Factor Reviews. 2004; 15:457-475) (17)

25 CONTROL LATRUNCULIN A Figure 1.4. The dynamic nature of the podosomes of actin rings Rhodamine actin was incorporated into saponin permeabi lized osteoclast like cells. In the control cells, the rhodami ne actin was quickly incorpor tated (within 10 minutes) into the actin rings of osteoclasts. In t he latrunculin A treated cells, which inhibits G-actin from polymerization, a complete loss of the actin ring was observed. (Hurst and Holli day, unpublished)

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26 ACTIN MERGE V-ATPase Figure 1.5. In unactivated osteoclasts, V-ATPase is not pres ent at the plasma membrane but is stored in cytoplasmic vesicles, but upon activation, it is transported via actin filaments to the ruffled membrane. Mouse marrow osteoclasts were loaded onto bovine cortical bone slices cultured for 2 days, and fixed and stained with anti-VATPase antibody and phalloid in. This micrograph is representative of an early resorptive os teoclast. The white arrow identifies a region where the V-ATPase has been trans ported to the ruffled membrane which is bounded by actin. The black arrow, below, identifies a unactivated region, where the V-ATPase and acti n are still found to be co-localized in cytoplasmic vesicles (Lee et al. J Biol Chem 1999; 274(41):29164-29171) (9)

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27 CHAPTER 2 ACTIN RELATED PROTEIN (ARP) 2/3 COMPLEX: AN ELEMENT OF ACTIN RINGS Introduction The Arp2/3 complex was or iginally identified by Machesky et al, 1994 (128) as a contaminant during affini ty chromatography of profilin from Acanthamoeba castellani Further studies have show n the Arp2/3 co mplex to be ubiquitous (129). It has been isolated and studied in detail from sources including human platelets, bovine br ain extract, Xenopus laevis and Sacchromyces cerevisiae (130-133). The Arp2/3 comp lex is a globular particle of 220 kD (134, 135) and is composed of seven subuni ts (131, 136-139), which have been highly conserved during evol ution (136). Arp2 and Arp3 are actin related proteins, sharing sequence hom ology with actin in the nucleotide and divalent cation binding domains (131). The other five subunits are novel (131, 137, 139). The subunits ar e present in stoichiometr ic amounts (131, 140). Two isoforms of both the Arp3 and p40 subunits have been identified ( 130, 133, 139, 141). The two isoforms of the Arp3 subun it, Arp3 and Arp3B, share 92% identity(139). Expression of the two isoforms di ffers with tissue (139). Arp3 is present ubiquitously, while Arp3B is found predominantly in th e brain, liver, muscle and pancreas (142). The two isoforms of the p40 subunit share only 68% sequence similarity (130, 133, 139, 141).

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28 The Arp2/3 complex is a key regul ator and nucleator of actin polymerization (32, 129). The Arp2/3 complex functions to stimulate actin polymerization at the barbed end of actin filaments, form a nucleation core to trigger actin polymerization de novo, and bi nd to the side of actin filaments where actin polymerization is triggered, resulti ng in the formation of an orthogonal actin network (134, 136, 137). Neither Arp2 no r Arp3 is able to independently induce polymerization of actin (133, 136). T he formation of a dimer between the two subunits in the complex is required to form the nucleation core to trigger polymerization of actin (Figur e 2.1) (138); this process is considered a possible rate limiting step (137, 138, 143, 144). The formation of the dimer is a result of activators such as the WASP family pr oteins, VASP via ActA, and cortactin (138, 143-146). Arp2/3 complex driven polymerization is th ought to be required for centrally-important cell processes including amoeboid movement and phagocytosis (147-150). The fa ct that the Arp2/3 complex is a central player in the actin-based motility of certain pat hogens has proven to be invaluable to understanding how Arp2/3 wo rks (130, 148-150). Activa tion of the Arp2/3 complex by WASP family members and sm all G-proteins results in actin polymerization resulting in the movem ent of bacterial pathogens such as Listeria, Shigella and Rickettsia as well as the enveloped vi rus vaccinia (129, 151-153). This motility actin polymerization that se rves as the basis for this movement results in an actin comet tail. This mo vement is involved in the spread of the pathogens from cell to cell (145, 149, 150). Reconstitution of actin-based

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29 motilities in vitro has been successful using F-acti n, the Arp2/3 complex, actin depolymerizing complex (ADF), and capping protein (154). The motility of this system proceeds at slow speeds; however, with the addition of Arp2/3 regulators, such as profilin, actinin, and VASP, there is an incr ease in motility (154). In this study, we examined the presence of the Ar p2/3 complex in osteoclasts and its localization during ost eoclastogenesis. In addition, we tested its requirement for actin ring formation. Materials and Methods Materials Anti-Arp2 and anti-Arp3 antibodies we re purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rhodamine labeled phalloidin was obtained from Sigma-Aldrich (St. Louis, MO). All CY2 and Texas Red-labeled secondary antibodies were obtained fr om Jackson-ImmunoResearch (West Grove, PA, USA). The expression ve ctor containing a RANKL [158-316] glutathione-S-transferase fusion protein construct was a kind gift of Dr. Beth S Lee (Ohio State University Columbus, OH, USA) Arp 2/3 purification The Arp2/3 complex was purified fr om outdated human platelets (Civitan Blood Bank, Gainesville, FL, USA) by a method previously described by Welch and Mitchison (155) based on conventional chromatography. The platelets were centrifuged at 160g for 15 minutes. The platelet pellet was resuspended in 20 volumes of wash buffer (20 mM PIPES, pH 6.8, 40mM KCL, 5 mM ethylenebis(oxyethylenenitrilo)tetraac etic acid (EGTA), 1 mM Et hylenediaminetetraacetic

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30 acid (EDTA) per volume of packed platelets and centrifuged at 2000g for 15 minutes. The wash was repeated two times. After the final spin, the pellet was resuspended in five volumes of wash bu ffer on ice for 10 minutes. An equal volume of lysis buffer (Wash buffer pl us 10 ug/ml leupeptin, pepstatin, and chymostatin (LPC protease inhibitors), 1 mM benzamidine, 1 mM phenylmethylsuflonyl fluoride (PMSF), 1% Triton X-100, and 0.05 mM adenosine triphosphate) was added on ice for 5 minute s. The lysate was centrifuged at 2000g for 2 minutes at 4oC to pellet the triton-insolubl e cytoskeleton. The pellet was resuspended in 5 volumes of resusp ension buffer (Wash buffer plus LPC protease inhibitors, 100mM sucrose, 0.05 mM ATP and 1 mM dithiothreitol (DTT)). The resuspended lysate was c entrifuged at 2000g fo r 2 minutes at 4oC. The pellet was gently resuspended in 10 volumes of low salt buffer (20 mM PIPES, pH 6.8, 10mM KCl, 5 mM EGTA, 1 mM EDTA, 1 mM DTT, LPC protease inhibitors) and repelleted by centrifugat ion at 2000g for 2 minutes. The pellet was resuspended in 5 volumes of extracti on buffer (20 mM PIPES, pH 6.8, 0.6 M KCl, 5 mM EGTA, 1 mM EDTA, 1 mM D TT, 0.2 mM ATP, LPC protease inhibitors). This suspension was hom ogenized for 2 minutes using a Teflon tissue homogenizer. The homogenate was incubated on ice for 30 minutes and then centrifuged at 25,000g for 15 minutes. The supernat ant was collected the first fraction of the cytoskeletal extract. The pellet was resuspended in 5 volumes of extraction buffer, and homogenized for 1 minute, followed by incubation on ice for 2 hours. This step was repeated two ti mes; after which, the homogenate was centrifuged at 25,000g for 15 minutes at 4oC. The supernatant was collected and

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31 added to the first fraction of the cytoskele tal extract. Figure 2.2 lane 1 shows the total protein extract from the human plat elets. ATP was added to a 5 mM final concentration and EGTA was ad ded to a 10 mM final concentration. The extract was incubated at 4oC for 16 hours. The extract wa s centrifuged at 25,000g for 15 minutes. The extract was desalted by us e of a 10 ml gel filtration column preequilibrated with Q-Buffe r A supplemented with 100 mM KCl (20 mM Tris, pH 8.0, 2 mM MgCl2, 5 mM EGTA, 1 mM EDTA, 0.5 mM DTT, 0.2 mM ATP, 2.5% v/v glycerol). The desalted extract was passed over a 5 ml-Hi-trap Q-Sepharose HP Column pre-equilibrated with Q Buffer A plus 100 mM KCl. The column was presaturated with ATP prior to loading the desalted extract. The Arp2/3 complex is isolated in the flow through fractions Figure 2.2 lane 2 shows the protein composition of the Q-S epharose flow through fraction. The flow-through fractions were pooled and the pH was adjust ed to pH 6.1 by the addition of MES, pH 6.1 to a final concentration of 40 mM. Glycerol to 10% v/v and LPC protease inhibitors were added and the KCl concentration was adjusted to 50 mM by the 1:2 dilution of sample to S-buffer A (20 mM 2-[N-Morpholino] ethanesulfonic acid (MES), pH 6.1, 2 mM MgCl2, 5 mM EGTA, 1 mM EDTA, 0.5 mM DTT, 0.2 mM ATP, 5-10% v/v glycerol). The diluted flow-through fractions were passed over a 1 ml Hi-trap SP-Sepharose HP column pr e-equilibrated with S-buffer plus 50 mM KCl at a rate of 0.5 ml/min. The column was washed with 10 volumes of S buffer with 50 mM KCl. The Arp2/ 3 complex was eluted with a linearly increasing gradient of KCl from 50 mM to 500 mM. The Arp2/3 complex eluted at 175-200 mM KCl. The peak fractions were pool ed and concentrated to 0.5 ml.

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32 The concentrated fractions we re loaded onto a Superose 6-HR 10/30 gel filtration column pre-equili brated with gel filtration bu ffer (20 mM MOPS, pH 7.0, 100 mM KCl, 2 mM MgCl2, 5 mM EGTA, 1 mM EDTA, 0.5 mM DTT, 0.2 mM ATP, 5-10% v/v glycerol). Fractions of 0.5 ml were collected and the Arp2/3 complex was the only detectable peak eluted from the column at A280. The fractions containing the purified Arp2/3 complex were pooled and concentrated using Centricon 30 concentrators. The protein was frozen in liquid nitrogen and stored at oC. Approximately 500 ug of protein was recove red from 10 ml of cytoskeletal extract (250 ml of plasma). Cell culture Osteoclasts were obtained from two s ources. Mouse marrow osteoclasts were grown from marrow derived from t he long bones of the hi nd legs of SwissWebster mice. The marrow cells were grown in -MEM medium with 10% fetal bovine serum (FBS) plus 10-8 M 1,25-dihydroxyvitamin D3 for a period of approximately seven days. Osteoclasts were also grown from the RAW 264.7 cell line, which is a mouse hematopoietic ce ll line. This protocol was approved by the University of Florida Institutional Animal Care and Usage Committee. RAW 264.7 cells were grown in Dulbecco s Modified Eagles Medium (DMEM) containing gentamicin and 10% FBS for 4 days with fresh media being added on day 2. On day four, the cells were detac hed by scraping, gently triturated and counted with a hemacytometer. The cell density is crucial for osteoclast differentiation. A cell c ount of 15,000-20,000 cells/cm2 was cultured with 50 ng/ml recombinant receptor activato r of nuclear factor kappa b ligand

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33 (RANKL)(amino acids 158-316)-GST for 4-5 da ys. With the ad dition of RANKL, the RAW 264.7 cells become large, multinucleated cells expressing characteristics of osteoclasts includi ng actin ring formation, expression of tartrate-resistant acid phosphatase activi ty and the ability to resorb bone. The osteoclasts and RAW 264.7 cell s were cultured in tiss ue-culture grade dishes. Once mature, the cells were scraped and r eplated on either glass coverslips or dentine bone slices. Western blot analysis with quantitation of Arp2/3 Anti-Arp2/3 antibodies were obtained from Santa Cruz Biotechnology Inc (Santa Cruz, CA). The Anti-Arp2 anti body was generated again st the carboxyl terminus of the Arp2 protein while the Anti-Arp3 antibody was generated against the amino terminus. The specificity of the antibodies was determined by Western Blot analysis, by probing the purified Arp2/3 complex (F igure 2.3A). RAW 264.7 cells were grown as previously described, plated on 6 well plates, and either left unstimulated or stimulated with RANKL. Ce ll lysates were collected from both the control and treated cells. Cells were washed twice with ice cold PBS and scraped from the plates. The cells were then detergent solubilized in 0.2% Triton X-100 in PBS. Equal am ounts of the lysates were separated by SDS-PAGE, followed by Western Transfer. The nitroc ellulose blots were then incubated with either anti-Arp3 or antiArp2 antibodies for one hour, washed three times, incubated with HRP conjugat ed secondary antibody, washed three times, and incubated with Super Signal Dura West Chemiluminescent Substrate (Pierce, Rockford, IL). The blots were then viewed on a Fluorochem 8000 (Alpha-

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34 Innotech, San Leandro, CA), and qua ntitation was performed by Spot Densitometry (Fluor-Phor Software, Al pha-Innotech, San Leandro, CA). The integrated density values (IDV) were obtained (white = 65535, black = 0). Background values were subtracted, and t he intensities were normalized against the value of actin in the sample. The values were then compared between stimulated and unstimulated cells. The st imulated and unstimulated values were statistically analyzed using the students t-test, with st atistical significance (p) being less than 0.05. Immunofluorescence Immunofluorescence was performed to vi sualize the distribution of the Arp2/3 complex in the resorptive osteocla st as well as its co-localization with actin. The marrow or RAW264.7-deriv ed osteoclasts were fixed in 2% formaldehyde in PBS on ice for 20 minutes The cells were then detergentpermeabilized by the addition of 0.2% Triton X-100 in PBS for 10 minutes, washed in PBS and blocked in PBS with 2% bovine serum albumin (BSA) for one hour. Cells were stained with rhodaminephalloidin, or antibodies recognizing Arp3 or Arp2 at a dilution of 1:100 in PBS Secondary antibodies were diluted according to manufacturers instructions. Osteoclasts were visualized using the MRC-1024 confocal laser scanning micr oscope and LaserSharp software (BioRad, Hercules, CA). Images were taken in sequential series to eliminate any overlap of emission and analyzed by confocal assistant software. Additional imm unofluorescence experimentation was performed to identify changes in the distribution of the Ar p2/3 complex when introduced to agents

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35 known to disrupt actin ring formation. Cell culture was perfo rmed as previously described. On day 6 of differentiation (ma ny large multinucleated cells present), wortmannin (100 nM), cytochalasin D (20 M) or echistatin (10 nM) were added to the cells and incubated for 10-30 minutes The cells were then fixed in 2% formaldehyde, solubilized in 0.2% Trit on X-100 in PBS and blocked in PBS with 2% BSA. Cells were stained with rhodamine-phalloidin, or antibodies recognizing Arp3 or Arp2 at a dilution of 1:100 in PBS. Secondary antibodies were diluted according to manufacture rs instructions. Osteoclasts were visualized using the M RC-1024 confocal laser scanning microscope and LaserSharp software (Bio-Rad, Hercules, CA ). Images were taken in sequential series to eliminate any overlap of emi ssion and analyzed by confocal assistant software. Polymerase chain reaction of the two isoforms of Arp3 To determine the redundancy of the Arp3 protein, RNA was extracted from RANKL differentiated RAW 264.7 cells as well as from unstimulated RAW 264.7 cells using RNAeasy Mini Kit (Qiagen, Valencia, CA) and quantified by spectrophotometer. The sequences for Arp3 and Arp3-beta were obtained from Gen Bank. Primers were designed as des cribed in Table 2.1. For standard RTPCR, 3 g of total RNA was annealed to an oligo-dt prim er and first strand cDNA synthesis was performed us ing Thermoscript RT-PCR System (Invitrogen, Carlsbad, CA) following manufacturers dire ctions. One-twentieth of the cDNA was subjected to amplification by P CR. PCR was performed under the following conditions: 95oC for 2 minutes, then 35 cycles of 90oC, 30 seconds; 58oC, 30

37 with DMEM supplemented with FBS and RANKL. No antibiotics were used. The cells were incubated for 24 hours at 37o C in a CO2 incubator; after which, the cells were fixed in 2% paraformaldehy de. Rhodamine phalloidin was used to visualize actin ring morphololgy. Only cells with uptake of the fluorescent oligomer were identified as having been transfected wit h the control or Arp2 siRNA. Morphological exam ination was performed using confocal microscopy. Mouse marrow osteoclasts were grown on tissue culture plates for 5 days and supplemented with calcitriol as described previously. The cells were then scraped and transfected as described fo r the RAW 264.7 cells, except MEM was used in place of DMEM. Cells we re analyzed as described above for RAW 264.7 cells. For assessment of prot ein expression, RANKL stimulated RAW 264.7 cells on 6 well pl ates were either not transfe cted or transfected using 7.5 U control or experimental siRNA combined with 10 ul Lipofectamine 2000 on day 5 of differentiation. Six hours after transfe ction, the media was replaced by DMEM with FBS and RANKL. The cells we re incubated for 30 hours at 37o C in a CO2 incubator. Cells were scraped and washed twice with PBS. The pellets were lysed using 250 ul of cell extraction bu ffer (BioSource International, Camarillo, CA, USA) supplemented with protease inhibitor cocktail (Sigma P2714) and phenylmethylsulfonyl fluoride (PMSF) for 30 minutes on ice with vortexing every 10 minutes. The extract was centrif uged for 10 minutes at 13,000 rpm at 4o C. Bradford assay was performed on the lysates. Equal c oncentrations of protein were separated by SDS-PAGE, followed by western transfer. The nitrocellulose blots were blocked in bl ocking buffer overnight and in cubated with bot h anti-Arp2

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38 and anti-actin antibodies for 2 hours. T he blots were washed and incubated with a horseradish peroxidase (HRP)-labeled secondary antibody for 1 hour, followed by incubation with a chemiluminescent su bstrate. The blots were visualized using an Alpha Innotech Fluorochem 8000. Quantitation was performed using densitometry measuring integr ated density values. Results Arp2 and Arp3 are upregulat ed during osteoclastogenesis After the purified Arp2/3 comp lex was isolated from platelets, the specificities of the anti-Arp2 and ant i-Arp3 antibodies we re determined by western blot analysis (Figure 2.3A). Bo th antibodies recognized their target proteins. When observing total protein leve ls, by western blot analysis, from nonstimulated RAW 264.7 cells and RAW 264.7 cells induced to differentiate into osteoclasts by treatment with RANKL, both Arp2 and Arp3 were upregulated approximately three-fold in response to RANKL stimulation (Figure 2.3B and 2.3C). Both isoforms of the Arp3 pr otein are present in osteoclasts The Arp3 protein has been identified in two different isoforms. By PCR analysis, both isoforms are expressed in t he activated osteocla st (Figure 2.4). This may allow for redundancy of the Arp3 protein, which would allow the maintenance of essential func tion of the Arp 2/3 protei n even if one isoform was mutated or lost.

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39 Expression of Arp2/3 complex in the actin ring The actin rings on osteoclasts of either glass coverslips or bone slices were stained with ant i-Arp3 and anti-Arp2 antibodies (Fig ure 2.5). In addition to actin ring staining, osteoclasts on covers lips often showed intense patches of Arp3-staining with little F-actin co-sta ining in the center of the cell. Confocal z-sections of actin rings of osteoclasts on coverslips and on resorbing bone slices revealed that Arp3 was present throughout the actin ring and was enriched, relative to F-actin, at the apical membrane, in proximity to the sealing zone. Figure 2.6 A and B show a pr ojection of 44 slices of the edge of a mouse marrow osteoclast on glass stained with anti-Arp3 (A) or phalloidin (B). These slices were stacked and digitally rotated 90o so that the apical surface was at the bottom and the basolateral at the top. Figure 2.6C is the rotated version of 2.6A and Figure 2.6E is the rotated versi on of 2.6B. Figure 2.6E is the merged image of Figures 2.6C and 2.6D, with the Arp3 staini ng pseudocolored green and phalloidin staining pseudocol ored red. Notice that Arp3 was enriched compared with F-actin at the apical boundary, and F-actin was re latively enriched near the basolateral boundary. Figures 2.6F and G show a proj ection through the actin ring of a resorbing osteoclast stained with anti-Ar p3 (F) or phalloidin (G). Figures 2.6H and 2.6I show a smaller portion of t he rings found in Figures 2.6F and 2.6G. The smaller section was rotated 90o so that the apical surfac e, which contacts bone, was down, and the basolateral su rface was at the top (Figur e 2.6J). Using a small section of the actin ring, the image was si mplified and more easily interpreted.

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40 Anti-Arp3 staining was pseudocolor ed green and phalloidin staining was pseudocolored red. Similar results we re observed as with the unactivated osteoclasts. Arp3 was enriched relative to F-actin at the apical boundary. Arp3 does not co-localize with the actin associated protein, vinculin Osteoclasts were co-stained with ano ther actin associated protein, vinculin. The vinculin staining (Figure 2.7) surrounded that of Arp3 with little colocalization occurring. Disruption of Arp3 distribut ion by chemical agents The distribution of the Arp2/3 complex was identified after disruption of the actin ring by the chemical agents, wort mannin, cytochalasin D and echistatin (Figure 2.8). Disruption of the actin ring occurred r egardless of the chemical agent used; however, the Arp2/ 3 complex continued to co -localize with actin in podosomes (Figure 2.9). Figure 2.10 quant itatively describes the effects of wortmannin and echistatin treatment on osteoc last-like cells on glass coverslips. Arp2 is required for actin ring formation Five siRNAs were generated against ta rgets in Arp2. Pr eliminary studies showed that one (19942) effectively knoc ked down Arp2 expression, whereas the others were ineffective. RAW 264.7 ce lls were stimulated with recombinant RANKL and transfected just as they began to fuse. Transfection efficiency was from 65 to 80% of the to tal giant cells, as judged by uptake of a fluorescent double-stranded oligomer. Western blot anal ysis (Figure 2.11) of osteoclasts 30 hours after transfection showed a 70% decr ease in the amount of Arp2 found in the total cell extract.

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41 Other RAW 264.7 osteoclast-like cells were fixed 30 hours after transfection with effective or ineffectiv e siRNAs. Both nontransfected cells or cells transfected with ineffective siRNAs showed normal actin rings (Figure 2.11). In contrast, fewer structures that look like podosomes were apparent in the knock down cells, and actin rings were rarely observed (less than 1% of controls). Typically F-actin was concentrated in centra l regions of giant ce lls in which Arp2 was knocked down. Mouse marrow osteoclasts were al so transfected with effective or ineffective siRNAs (Figure 2.13). Transfe ction efficiency was very low, but a few transfected osteoclasts were identifi ed based on the entry of the fluorescent double-stranded oligomer. Osteoclast tr ansfection with 19942 did not have actin rings after 30 hours, whereas the majority of the osteoclasts transfected with the ineffective control did show actin rings. This was true for both activated and inactivated osteoclasts. Discussion These studies demonstrate for the firs t time that the Arp2/3 complex is a component of the actin ring of osteoclast s and is required for its formation. The Arp2/3 complex was upregulated three-fold during differentiation. This is consistent with the Arp2/3 playing a role in actin ring formation, specialized structures specific to ost eoclasts. The Arp2/3 comple x is abundant in actin rings, co-localizes with the actin core of podosomes and is enric hed at the apical boundary near where the osteocla sts contact the substrate. Vinculin, a focal adhesion protein, was enriched at the apical border of ac tin rings but did not co-

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42 localize with actin or the Arp2/3 comple x but rather surrounded them in a cloud, which is consistent with current studies (33). The organization of podos omes in the actin rings of osteoclasts has been shown to be disrupted by the addition of ch emical agents such as wortmannin, echistatin and cytochalasin D. Cytochalasin D is a fungal toxin that reduces actin polymerization by inhibiting G-actin and is known to disrupt actin ring formation in the osteoclast (156, 157). The actin fibers of podosomes depolymerize as the effective concentration of G-actin becomes limiting (156, 157). Wortmannin is a fungal toxin and functions as a selective inhibitor of PI3 Kinase activity (158). Echistatin is a snake venom to xin and inhibits the integrin, v3 (159, 123). In osteoclasts, echistatin causes a di sruption of the sealing zone and an internalization of integrin s from the basolateral membranes to intracellular vesicles. The treated osteoclasts tend to round up and collapse. Although the osteoclasts are still adherent to bone, osteoc lastic resorptive ability is severely reduced as is seen by a reduction in resorptive pit number and size. Regardless of the type of inhibition, disruption of t he actin ring occurs but with a continuous co-localization of the Arp2/3 comple x with the podosomal core. These data support high integrity of the podosom al core. It has become clear that much of t he actin filament dynamics in cells depends on the Arp2/3 complex (160). Ac tivated Arp2/3 comp lex interacts with actin monomers to promote f ilament assembly. Activation occurs in response to interactions with accessory proteins that are in turn activated in response to signal transduction. Recent data indica te that actin treadmills rapidly through

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43 podosomes, entering apically and removed basolaterally (Figure 2.15) (161). The plasma membrane is pushed forward by this actin polymerization until capping of the barbed end occurs. As the filaments age, the ATP bound to each subunit is hydrolyzed, with slow dissociation of the -phosphate. ADF/cofilin cause severing of actin filaments and t he dissociating of AD Pactin (161, 162). The exchange of ADP for ATP is catalyzed by profilin, and a regeneration of the pool of profilactin is ava ilable for the next generation of filaments (162). This mechanism suggests a role for the Arp2/3 complex. In addition, the enrichment of the Arp2/3 complex at t he apical boundary of the podosomes of actin rings that we observed is consistent with the Arp2/3 complex playing a role in the entry of actin monomers into the actin ring filam ents. The true functi on of the treadmilling is not currently known; however, it is plausible that the podosomes may be exerting force on the plasma membrane, caus ing it to conform to bone (160). It is known that actin polymerization can pr oduce protrusive forces required for cell crawling as well as the intracellula r propulsion of microbial pathogens and organelles. An important example of this force generation via actin polymerization occurs is in the propulsion of Listeria monocytogenes. Loisel et al. (154) have shown the reconsti tution of sustained movement in Shigella and Listeria with the addition of pur ified Arp2/3 complex, acti n, actin depolymerizing protein (cofilin), and capping protein. As the Arp2/3 co mplex is a known central player in the actin-based motility of cert ain pathogens, this same force generation may be within the realm of the Arp2/3 comp lex in the actin ring of osteoclasts (144-146).

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44 In osteoclasts, gelsolin has been implicated in triggering actin ring formation (44, 163). This could potentiall y be accomplished by cleaving existing filaments and uncapping barbe d ends in a regulated m anner (164). Moreover, the gelsolin knockout mouse is mildly osteopetrotic, suggesting a role for gelsolin in bone resorption (165). Howeve r, the mildness of the osteopetrosis suggests other mechanisms contribute to the cytoskeletal dynamics required for bone resorption (166, 167). A strong po ssibility may be coordination between gelsolin and the Arp2/3 co mplex. A recent m odel describing podosomes suggests a balance of actin polymerization, which, based on our re sults, is likely regulated by the Arp2/3 comple x, and filament cleavage, by proteins like gelsolin (33). This balance could account for t he structure and dynamics of podosomes. In summary, the Arp2/3 complex is pr esent in the podosomal structures of the actin rings of osteoclasts. Knock down of Arp2 using siRNA shows that the Arp2/3 complex is required for actin ring formation. These data suggest that the Arp2/3 complex plays a role in osteocla stic bone resorption and may provide a target for therapeutic agents designed to limit the activi ty of osteoclasts.

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45 Figure 2.1. The Arp 2/3 complex. A) Crystal structure of the 7 subunits of the Arp2/3 complex. B) The Arp2/3 complex remains in an inactive conformation. Upon activation by WASP family member s, the Arp2 and Arp3 subunits undergo a conformational change and allow the complex to beco me active and participate in actin polymerization. (Robinson et al. Science. 2001; 294:1679-1684) (138)

47 0 5000 10000 15000 20000 25000 30000 35000 Figure 2.3. Arp2 and Arp3 are upregulated during osteoclastogenesis (A) Human platelet Arp2/3 co mplex was subjected to SDS-PAGE, blotted to nitrocellulose, and probed with antibodies against Arp3 and Arp2, and the bound antibody was detected by chemiluminesc ence. B) RAW 264.7 cells were cultures with (black bars) or without (w hite bars) RANKL. Total protein was extracted and equal amounts of protein were loaded and separated by SDSPAGE and transferred to nitrocellulose and probed with anti-actin, anti-Arp2 and anti-Arp3 antibodies. Arp2 and Arp3 expression was upregulated during osteoclastogenesis compared with actin. C) Quantit ation of four independent blots confirmed upregulation of Arp2 and Arp3 as osteoclasts differentiated. Error bars represent standard error. p < 0.05 by students t-test. A r p 3 Ar p 2 *

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48 Arp3b Arp3 GAPDH S U S U S U Figure 2.4. The two isoforms of Ar p3, Arp3 and Arp3-beta, are present in unactivated and activated osteoclasts. RAW 264.7 cells were cultured with (stimulated) or without (unstimulated) RANKL. Cells were harvested and RNA was obtained using RNAeasy Mini Kit (Q iagen, Valencia, CA). RT-PCR was performed using primers specific to Ar p3 and Arp3-beta. Both Arp3 and Arp3beta were present and are upregulated in response to RANKL stimulation. Figure 2.5. Arp2/3 co mplex is present in the actin rings of osteoclasts. Mouse marrow osteoclasts were loaded onto bovine cortical bone slices (A-C) or glass coverslips (D-E), cultured for 2 days, and fixed and stai ned with anti-Arp3 antibody (A and D) and phalloidin (B and E). Im ages were merged (C and F), with Arp3 staining pseudocolor ed green and phalloidin pseudocolored red. Colocalization of the two is yellow. A-C) A projection of 15 confocal slices (0.5 m) is shown. The arrow indicated the acti n ring. The green stai ning of the nuclei was the result of cross reactivity by the secondary antibody. Note the yellow staining of the actin ring in the merged image indicating co-loca lization. D-F) This is an image of a single optical section (0.5 m) of a mouse marrow osteoclast on a glass coverslip. The sma ll arrow points to Arp2/3-rich spots; the large arrow identifies the actin rings. The size bar is equivalent to 5 m in A-C and 25 m in D-F.

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49 Figure 2.6. Arp2/3 complex is enriched rela tive to F-actin near the sealing zone. A and B) A projection of the edge of an os teoclast on a coverslip is shown, stained with (A) anti-Arp3 or (B) phalloidin C-E) The images in A and B were computer rotated 90o to examine the cell in side vi ew. The apical side is down. The podosomal nature of the ring is read ily apparent. As shown by the arrows, Arp3 (pseudocolored green) was enriched near the apic al surface (the contact area with the coverslip), whereas micr ofilaments (pseudocolored red) were enriched at the basolateral bou ndary of the actin ring. Areas of co-localization are yellow. F and G) The image of a resorbing osteoclast on a bone slice is shown. H and I) A section of the actin ring is identified from F and G. J) The images in H and I were then merged and rotated 90o so that the apical surface was down. Arp3 is pseudocol ored green and phalloidin is red. As observed in the osteoclast on a glass coverslip, Ar p3 is enriched near the apical boundary near the sealing zone (arrow). The size bar is 10 m in A and B; 5 m in C-I, and 2 m in J.

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50 Figure 2.7. Arp2/3 does not co-localize with vinculin in actin rings. RAW 264.7 cells were stimulated with RANKL to differentiate into osteoclast-like cells and fixed and stained with either anti-Arp3 or ant i-vinculin. The images were merged. A) Image of actin ring st ained with anti-Arp3 and pseudocolored red. B) Image of actin ring stained wit h anti-vinculin and pseudocol ored green. C) Merged image of A and B. Note there is little co-localization between Arp3 and vinculin. The size bar is 3 m.

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51 Actin Arp3 Control Cytochalasin D Echistatin Wortmannin Figure 2.8. Treatment with the chemical agents, cytoc halasin D, echistatin and wortmannin, cause a disrupti on of the actin rings of osteoclasts. Mouse marrow osteoclasts were loaded onto bovine cort ical bone slices or glass coverslips, cultured for 2 days, and eit her untreated or treated with with cytochalasin D, echistatin or wortmannin for 30 minut es and fixed and stained with anti-Arp3 antibody and phalloidin. Note the disruption of the acti n ring in all cells but colocalization of the Arp2/3 comple x with actin remains stable.

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52 ARP3 AC TIN MERGE Figure 2.9. Arp2/3 remains co-localiz ed in the actin based podosomal core regardless of actin ring disruption by wortmannin. RAW 264.7 cells were cultured with RANKL until oste oclast-like cells were observed. The cells were then treated with 100 nM wortma nnin for 15 mintues, afte r which they were fixed and stained with either rhodam ine phalloidin or anti-Arp3 antibody. Although actin ring structure has been disrupt ed, Arp3 continues to co-localize with actin in the podosomal core. Figure 2.10. Wortmannin and echistatin treatment of osteoclasts results in a decrease in the number of actin rings. Ac tin rings were counted after either no treatment or treatment with wortmannin or echistatin A significant decrease in actin rings, more than 90%, was observed afte r treatment with either inhibitor.

54 FITC-OLIGOMER TRITC-PHALLOIDIN NO TREATMENT 19941 19942 Figure 2.12. Actin rings are disrupted in Arp2 knockdown Untransfected RAW 264.7 osteoclast-like cells or osteoclast-like cells transfected with ineffective siRNA (19941) or effective siRNA ( 19942) were fixed after 30 hours and examined for the presence of fluorescent ol igo marker of transfection (left panels) or F-actin by staining with phalloid in (right panels). The photographs are representative cells. The effective siRNA disrupted the ability of the osteoclasts to form actin rings. The size bar equals 25 m.

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55 Figure 2.13. Actin rings ar e disrupted in marrow osteocla sts on coverslips or on bone slices by siRNA directed against Arp2 Mouse marrow in tissue culture plates was stimulated with calcitriol for 5 days to produce osteoclasts. These were scraped and loaded onto coverslips (A-D) or bone slices (E-H) and transfected with (A, B, G, and H) 19942 or (C-F) 19941. The cells were stained with phalloidin (B, D, E, and G) or the fluorescent olig omer (A, C, F, and H) was detected. Note that in osteoclasts transfected with the effective siRNA (19942), no actin rings were present. In cells transfected with the ineffective control siRNA (19941), actin rings appeared normal. Standard bar in D is for A-D and represents 10 m. Standard bar in H is for E-H and represents 10 m.

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56 Figure 2.14. Experimental siRNA reduces the number of actin rings on coverslips by over 95%. RAW 264.7 osteoc last-like cells or os teoclast-like cells transfected with no siRNA, ineffective si RNA (19941) or effective siRNA (19942) were fixed after 30 hours and examined fo r the presence of fluorescent oligo marker of transfection. The actin rings of the cells with the ma rker of transfection present were counted to quantify changes in the number of actin rings formed. There was a significant decrease in the num ber of actin rings after treatment with effective siRNA. Error bars represent stan dard error. p < 0.05 by students ttest. *

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57 Figure 2.15. Dendrit ic Nucleation Model. Upon activation of WASP/Scar family proteins, the Arp2/3 complex is activated, resulting in actin polymerization and side-branching of new f ilaments on existing filaments. As the filaments elongate, they push the membrane forward. Profilac tin is required for filament elongation at the barbed ends and may be localized to this region by VASP. (ATP-actin white; ADP-P-actin orange; AD P-actin red; profilin black) (Blanchoin L. et al. Nature. 2000;404:1007-1011) (37)

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58 Table 2.1. PCR Primers Us ed for Identification of Ar p3 Isoforms. The sequences of primers used for PCR as well as their positions numbered relative to the AUG start site and the expected product size. All primers were designed against murine sequences. RT-PCR Target Position of Primers Size of Product Sequence of Primers (5-3) 750-769 AGAGCACCAGAGAGAGCAGA Arp3 921-940 191 bp CACACCACACGGCTACTACA 380-403 CCATGTTTGTGATGGGTGTGAACC GAPDH (Control) 1068-1091 711 bp TGTGAGGGAGATGCTCAGTGTTGG \

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59 CHAPTER 3 THE ARP2/3 COMPLEX: A POSSIBLE LINK IN THE TRANSLOCATION OF V-ATPASE TO AND FROM THE RUFFLED MEMBRANE Introduction V-ATPase plays a vital role in the osteoclast as it is responsible for acidification of the extrac ellular compartment segregat ed by the osteoclast and subsequent demineralization of the bone mi neral (11, 12). Mutations in the V1 subunit B1 result in distal renal tubul ar acidosis accompanied by osteopetrosis (64). In addition, recessive osteopetrosis, with deficient acid secretion, is caused by mutations in the V0 domain or in the chloride channel (64). The vacuolar proton ATPase is composed of 13 or mo re different proteins and over 20 subunits and consists of two major functional domains, V1 and Vo (Figure 3.1) (11-170). The V1 domain, a peripherally loca ted cytoplasmic section, contains at least eight different subunits (A-H) and contains three catalytic sites for ATP hydrolysis (168). These sites ar e formed from the A and B subunits (11, 168). The Vo domain, a proton channel, is co mposed of at least 5 subunits and allows for proton translocation across the ruffled membrane (168). V-ATPase is present in osteoclast precursors at high levels (171); but upon osteoclastogenesis, the levels of V-ATPase increase significantly and

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60 isoforms selective to the osteoclast are expressed (171, 172). Prior to activation of the osteoclast, the V-ATPa se is stored in intracellula r cytoplasmic vesicles (23, 50). As the cell is activated, V-ATPase binds to actin and is transported to the ruffled membrane, a specialized region of the plasma membrane. Once a resorption cycle has been completed, the VATPase is internalized into the cytosol (173). V-ATPase binding to F-actin has been identified with the F-actin binding site localized to a profilin-like domain in subunit B (11). This domain is localized to amino acids 23-67 in the B1 subunit and bi nding is in a direct 1:1 relationship (174). Since there are three B subunits, there are at least three actin binding sites present on the V-ATPase, and two more may be associated with the C subunit as it has also been shown to bind acti n (175). It is of not e that the levels of actin bound to V-ATPase fluctuate with t he resorptive state of the osteoclast. Binding of F-actin to V-ATPase appears to be physiologically controlled with evidence supporting signaling through v3 and PI3K activity (12, 52, 163, 175177). During translocation of the V-ATPase to and from the ruffled membrane, F-actin and V-ATPase are components of discrete structures termed podosomes (178). There are several lines of ev idence supporting the dependency of the cytoskeleton for transportation of V-ATPa se to and from the ruffled membrane. The grey lethal mutation (gl), which caus es osteopetrosis, results in defective cytoskeletal organization (179). In the majo rity of cases, a mutation is found in the gene, TCIRG1, which encodes the a3 s ubunit of the osteoclast V-ATPase

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61 (179). Mutations of this protein may prohibit the V-ATPase from assembling which would be consistent with the lack of ruffled border formation and improper and disorganized localization of V-ATPa se (180-182). In addition, the oc/oc osteosclerotic mouse shows a lack of association between the cytoskeleton and V-ATPase, hindering the localization of V-ATPase to the ruffled membrane (180182). This mouse is characterized by extensive bone deformities (180-182). These data support the hypothesis that the detergent insoluble cytoskeleton plays a key role in transportation of t he V-ATPase to the ruffled membrane. As previously stated, t he Arp2/3 complex is a cent ral player in the actinbased motility of certain pathogens (144147). The Arp2/3 complex has been shown to co-localize with actin in the ac tin ring and as a vital component of the actin ring of osteoclasts. In addition, the Arp2/3 complex responds by various proteins, such as cortactin and VASP, which are members of various signal transduction pathways. From this interact ion with actin dynamics, its ability to be regulated by signal transduction me chanisms, and its sequence homology with actin, it might be hypothesized that t he Arp2/3 complex may bind V-ATPase, as actin does, and function as a possible player in the transportation of V-ATPase to and from the ruffled membrane. In this study, we tested for an a ssociation between V-ATPase and the Arp2/3 complex. Since no associatio n could be determined, other potential VATPase binding partners were identified.

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62 Materials and Methods V-ATPase/Arp2/3 binding assay To determine if the Arp2/3 complex bi nds to V-ATPase, a protein binding assay was performed. Twenty five l of a maltose binding protein (MBP) -B1 fusion protein (B1-109) was incubated with 25 l of purified Arp2/3 complex for 1 hour. Amylose beads, which are an affinity matrix used to isolate proteins fused to MBP, were prepared by sequential washes in column buffer followed by Fbuffer. The amylose beads (25 l) were then added to t he Arp2/3-fusion protein mixture and incubated for 30 minutes. The solution was centrifuged at 13,000 rpm for 2 minutes. The supernatant was collected, and the beads were washed with F-buffer. This was repeated three times. The beads were then incubated with 25 l of 100 mM maltose for 10 minutes and eluted by centrifugation. The supernatant was separated by SDS-PAG E and stained with Coomasie Blue. Immunoprecipitation was performed to id entify binding of Arp2/3 with VATPase. The MBP-tagged B1 fusion protei n was incubated with purified Arp2/3 complex and protein G beads (t o allow for clearance of any non-specific binding). The mixture was centrifuged and the supernat ant collected. Anti-maltose binding protein antibody was incubated with the supernatant for 30 minutes. Protein G beads were added and incubated for 10 minut es. The mixture was centrifuged and the supernatant collected (to determine in which frac tion the original sample was). The pellet was washed three time s. The pellet wa s incubated with SDS and centrifuged at 13,000 rpm for 2 minut es. The supernatant was then separated by SDS-PAGE follow ed by western transfer. The nitrocellulose blots

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63 were then incubated with ant i-Arp2 antibodies for one hour washed three times, incubated with anti-goat HRP conjugated secondary antibody, washed three times, and incubated with Super Signal Du ra West Chemiluminescent Substrate (Pierce, Rockford, IL). The blots were then viewed on a Fluorochem 8000 (Alpha-Innotech, San Leandro, CA). PCR to identify other actin associated proteins involved in V-ATPase translocation and actin ring dynamics To identify other key proteins invo lved in osteoclastogenesis, RNA was extracted from RANKL di fferentiated RAW 264.7 cells as well as from unstimulated RAW 264.7 cells using RN Aeasy Mini Kit and quantified by spectrophotometer. The sequences fo r WASP, n-WASP, VASP, Cortactin, and Arp3 were obtained from Gen Bank. Primers were desig ned as described in Table 3.1. For standard RT-PCR, 3 g of total RNA was annealed to an oligo-dt primer and first strand cDNA synthesis was performed using Thermoscript RTPCR System (Invitrogen, Ca rlsbad, CA) following manufacturers directions. One-twentieth of the cDNA was subject ed to amplification by PCR using the primers listed in Table 3.1. PCR was performed under the follo wing conditions: 95oC for 2 minutes, then 35 cycles of 90oC, 30 seconds; 58oC, 30 seconds; 72oC, 30 seconds. One-half of the PCR pr oduct was separated on 0.5% agarose gel with ethidium bromide st aining for 1 hour. Images were detected using UV transillumination on a Fluorochem 8000 (Alpha-Innotech, San Leandro, CA).

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64 Immunoprecipitation with the B subuni t of V-ATPase suggests a possible direct linkage between VASP and V-ATPase. To identify possible bindi ng partners with the B2 s ubunit of V-ATPase, cell lysates were extracted from RANKL stimulated RAW 264.7 cells. The cell lysates were subjected to high speed ce ntrifugation to pellet actin and to avoid the presence of actin fila ment complexes in the im munoprecipitate. The B2 antibody was biotinylated using EZ-link Su lfo-NHS-LC-biotinylat ion kit (Pierce, Rockford, IL). The lysates were incubat ed with either B2-biotinylated or B2 antibody. The B2 (non-biotinylated) anti body was used as a c ontrol. Complexes were pulled down with streptav idin agarose, which affini ty purifies biotin labeled proteins. The agarose was washed and eluted with loading buffer. The elution was separated by SDS-PAGE and western tr ansfer. The nitrocellulose blots were then probed with antibodies direct ed against various actin associated proteins such as N-WASP, cortactin, VASP, WASP, and Arp3. The blots were washed and incubated with secondary antibodies and incubated with Super Signal Dura West Chemilumi nescent Substrate (Pierce, Rockford, IL). The blots were then viewed on a Fluorochem 8000 (Alpha-Innotech, San Leandro, CA). Results The B1 (1-106) subunit of V-ATPase does not bind purified Arp2/3 complex. Purified Arp2/3 complex and the B1(1106) maltose binding protein fusion protein, which contains the actin binding site, were incubated together. After being separated on amylose resin and elut ed with maltose, the elution was separated by SDS-PAGE and We stern transfer. The blots were then probed with

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65 either anti-B1 or anti-Ar p3 antibody. Only the B1 subunit was pulled down, suggesting that the Arp2/3 complex does not bind to V-ATPase in the actin binding region (Figure 3.2 and 3.3). Cortactin is preferentially upregulat ed at the transcriptional level during osteoclastogenesis To identify other actin associated proteins involved in V-ATPase translocation and actin ring dynamics, PCR was performed using primers to detect changes in gene expression in severa l actin-associated proteins during osteoclastogenesis. Unlike the other proteins tested, cortactin mRNA was the only gene preferentially upreg ulated during osteoclasto genesis, with a complete lack of detection prior to treatment of RAW 264.7 cells with RANK -L (Figure 3.4). This was expected based on a previ ous publication which identified an upregulation of cortactin prot ein in chicken osteoclasts. These data identify upregulation occurs at t he transcriptional level. Vasodilator stimulated phosphoprotein (VASP) is identified to have a possible interaction with V-ATPase. A signal transduction assay was peformed using a standard array by Hypromatrix (work done by Sandra Vergara). The me mbrane was incubated with RANKL-induced RAW 264.7 whole cell extract. The membrane was then incubated with a biotinylat ed-B2 antibody, washed and labeled with a secondary antibody. Chemiluminescent substrate was applied and the membrane was viewed by a Fluorochem 8000. Among 29 responsive proteins, vasodilator

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66 stimulated phosphoprotein was identified as having an interaction with the B2 subunit (Figure 3.5). Immunoprecipitation with the B subuni t of V-ATPase suggests a possible direct linkage between VASP and V-ATPase. To identify possible bi nding partners wit h V-ATPase, cell lysates were extracted from RANKL stim ulated RAW 264.7 cells. The cell lysates were subjected to high speed centrifugation to remove any contamination by actin complexes in the immunoprecipitate. T he lysates were incubated with either biotinylated-B2 or non-biotinylated B2 antibody. Complexes were pulled down with streptavidin agarose, to isolate any protein complexes bound to the biotinylated antibody. T he non-biotinylated B2 antibody was used as a control. Efforts to pull down cortac tin in immunoprecipitations of V-ATPase were not successful; and of all the proteins tested, VASP was identified to form a complex with the B2 subunit of V-ATPase (Fi gure 3.6), suggesting a potential complex that includes VASP, cortactin and V-ATPase. Discussion The actin binding site on V-ATPase has been identified to amino acid sequence 23-67 of the B1 subunit of th e V-ATPase (183). Based on the sequence homology between actin and the Ar p2/3 complex, we hypothesized that V-ATPase might bind the Arp2/3 co mplex. Experiments with both binding assays and immunoprecipitatio n experiments with the B1 fusion protein failed to show a direct linkage between V-ATPase and the Arp2/3 complex. However, this result does not confirm an absence of a direct interaction between the two

PAGE 78

67 proteins. Binding of t he Arp2/3 complex may occur through a different amino acid sequence than that of the fusion protein or the Arp2/3 complex may not be in the correct structural conformation to bind to the V-ATPase in the performed experiments. Isolation of purified V-ATPase was attempted to determine binding with the Arp2/3 complex but has not been successful thus far. As identification of a direct inte raction between V-ATPase with Arp2/3 could not be established, re search focused on the identifi cation of other proteins which could play pivotal roles in osteoc last function. Se mi-quantitative PCR analysis of several actin related proteins was performed to determine if there were any changes during osteoclastogenesis. Cortactin was identified as being preferentially upregulated dur ing osteoclastogenesis at the transcriptional level (184), indicating a possible key role in ac tin ring formation or translocation of VATPase to the ruffled membrane. This finding is not surprising as previous research in chicken osteoclasts has shown the cortactin upregulation at the protein level (184); however, our findings identify for the first time that the upregulation occurs at a transcriptional le vel. Cortactin is involved in the activation and stabilization of actin based net works, inhibiting their disassembly (135, 185-187). Cortactin ca n bind and activate the Arp2/3 complex through binding the Arp3 subunit (186, 187). Cortactin, n-WASp, and Arp2/3 form a synergistic, ternary complex to initiate actin polymerizat ion (186, 188). Although no additional proteins were found to have significant differences in levels of mRNA before and after osteoclastogenesis real-time PCR woul d be of value in

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68 determining minor variations in mRNA co ncentration not detectable by traditional PCR. In addition to cortactin, we sought to identify other actin binding proteins that could have a possible interacti on with V-ATPase. A signal transduction antibody array was performed by Sandra Vergara (University of Florida, Gainesville, FL) to determine possible in teractions between signal transduction proteins and V-ATPase from RANK-L induced RAW264.7 whole cell extracts. The results from this array indicated t hat Vasodilator Stimul ated Phosphoprotein might be linked with V-ATPase. Further immunoprecipitation experiments show that VASP is pulled down in a complex wi th the B2 subunit of V-ATPase. VASP plays a key role in actin based motility and is localized predominantly at focal adhesions, cell/cell contacts and regions of highly dynamic actin reorganizations such as podosomes (151, 185). VASP can bi nd directly to G-actin and F-actin as well as recruit profilactin complexes to the site of actin polymerization. In addition, VASP is known to enhance Arp 2/3 activity and prevent capping proteins. VASP is phosphorylated in re sponse to protein kinase A (PKA) and protein kinase G (PKG) (189, 190). The ability of VASP to be phosphorylated allows it to be both a positive and negativ e regulator of actin polymerization. Calcitonin induces alterations in the cytoskeleton of the osteoclast through the protein kinase A pathway (191, 192). It is plausible that the disruption of the actin cytoskeleton by calcitonin could be mediated by VASP. Phosphorylation of VASP has also been shown to diminish F-actin binding, suppressing actin nucleation as well as inhibi ting Arp2/3 triggered actin poly merization; thus, it can

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69 be a negative regulator of actin polymeri zation (185). Thus, VASP may play an important role in the regul ation of the translocation of V-ATPase to and from the plasma membrane. In summary, the Arp2/3 complex di d not bind the same amino acid sequence of the B1 subunit of V-ATPase as did actin. Further studies are required to determine if binding exists at another sequence. Two additional proteins, cortactin and VASP, were identif ied as having possible key roles in osteoclast function. Cortactin was f ound to be preferenti ally upregulated in response to RANKL stimulation while VASP was found to associate with the B2 subunit, either directly or indirectly th rough other V-ATPase su bunits or other VATPase bound proteins.

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70 Figure 3.1. The st ructure of V-ATPase The vacuolar proton ATPase is composed of 13 or more diffe rent proteins and over 20 subunits and consists of two major functional domains, V1 and Vo. The V1 domain, a peripherally located cytoplasmic section, contains at least eight different subunits (A-H) and contains three catalytic sites for ATP hydrolysis. These sites are formed from the A and B subunits. The Vo domain, a proton channel, is co mposed of at least 5 subunits and allows for proton translocation acro ss the ruffled membrane. (Sun-Wada et al. Biochimica et Biophysica Acta. 2004; 1658: 106-114) (168)

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71 B1 (1-106) Puri fied Subunit of Arp2/3 V-ATPase Co mplex IP: Amylose B1 Figure 3.2. The B1 (1-106) fusion prot ein of V-ATPase and the Arp 2/3 complex do not show a direct interaction by bi nding assay. The B1-MBP fusion protein and the Arp2/3 complex were incubated t ogether. The sample was then run on amylose resin to bind the maltose binding protein. The column was then eluted with maltose. The samples were s eparated by SDS-PAGE and stained with Coomasie. The B1 subunit was pulled do wn in the amylose column but Arp3 was not, indicating a lack of binding between the two proteins.

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72 IP: MBP IP: MBP Probe: B1 Probe: B1 Probe: Arp3 Probe: Arp3 Figure 3.3. The B1 (1-106) fusion prot ein of V-ATPase and the Arp 2/3 complex do not show a direct interaction by immunoprecipitat ion of B1 subunit. The B1MBP fusion protein and the Arp2/3 comp lex were incubated together. The sample was then incubated wit h a maltose binding protei n antibody. The sample was then immunoprecipitated wit h protein G beads which bind the antibody. The beads were washed and eluted with sodium dodecyl sulfate. The elution was then probed using the B1 or Arp3 antibodies. B1 was pul led down by the protein G beads but Arp3 was not, indicating a lack of binding between the two proteins.

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73 Stimulat ed Unstimulated Cortactin WASP N-WASP VASP Arp3 GAPDH Figure 3.4. Cortactin is preferentially upregulated during osteoclastogenesis as identified by PCR. RAW 264.7 cells were cultured with (stimulated) or without (unstimulated) RANKL. Cells were harvested and RNA was obtained using RNAeasy Mini Kit. RT-PCR was performed us ing primers specific to cortactin, WASP, N-WASP, VASP and G APDH (control). Cortactin was the only actinassociated protein preferent ially upregulated in response to osteoclastogenesis.

75 B2 Biotin B2 IP: B2 subunit Streptav idin VASP Figure 3.6. Immunoprecipi tation experiments with t he B subunit of V-ATPase Suggests a Possible Direct Linkage between VASP and V-ATPase. RANKL stimulated RAW 264.7 cell lysates were incubated with biotinylated B2 antibody, pulled down on streptavidin agarose, separated by SDSPAGE and western transfer, and probed wit h the antibodies of various ac tin related proteins. Of all the proteins tested, only VASP was pulled down in complex with the B2 subunit of the V-ATPase.

77 CHAPTER 4 THE ROLE OF CORTACTIN IN OSTEOCLASTOGENESIS Introduction Cortactin is a monomeric, long, flexib le protein (186) with a multidomain structure consisting of an acidic domain at the amino terminus, followed by 6 and 1/2 tandemly repeated 37 ami no acid segments, a helical region, a proline rich region, and a Src homology 3 (SH3) domai n at the carboxyl terminus (135, 136, 186). The multidomain struct ure of cortactin allows a multitude of interactions. Cortactin binds directly to F-actin th rough sequences in the tandem region while binding to the Arp2/3 comp lex occurs at the amino terminus (136, 186, 188). Various signaling proteins bind the c-te rminal proline rich and SH3 domains (135, 185, 188). Cortactin is a physiologically significant substrate for tyrosine phosphorylation by src kinases (135). This is important because actin ring formation requires the activity of pp60c-src ( 193, 194). Mutations in c-src in mice results in osteopetrosis and failure of podosome formation (19). Faciogenital dysplasia protein 1 (Fgd1), a CDC42 guani ne nucleotide exchange factor, also binds the SH3 domain of cortactin ( 195). This association allows proper localization of Fgd1 to the actin cyto skeleton (196). Mutations in Fgd1 are implicated in the human disease faciogeni tal dysplasia (197, 198). The pathology of this disorder includes bone abnormalities.

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78 Cortactin is involved in the activation and stabilization of actin based networks (185, 186). Initiall y, the role of cortactin was hypothesized as a result of its localization to the same regions as Arp2/3 and n-WASP in vesicles, podosomes, and the actin based rocket tails of Listeria (135, 186, 188, 199). The function of cortactin as a regulator of the Arp2/3 comp lex is two fold. First, cortactin can bind and activate the Ar p2/3 complex through binding the Arp3 subunit (186, 188), although its activation potent ial is four to five fold lower than that of the WASP fam ily proteins (135, 187). Second, cortactin stabilizes Arp2/3 induced branched actin networks, inhibiting their disassembly (135, 187, 200). Recent studies suggest that cortactin, N-WASP, and Arp2/3 form a synergistic, ternary complex to initiate actin polym erization as depicted in Figure 4.1 (186, 188). In this model, N-WASP activates nucleation by interacting with F-actin and the Arp2 and p40 subunits while cortactin stabilizes the branching points by binding to F-actin and the Ar p3 subunit (135, 187, 200). Cortactins main role may invo lve the carboxy terminal SH3 domain. This domain allows interactions with various signaling molecules, including src kinases (186, 200). The tyrosine phosphoryl ation of cortactin occurs in response to integrin ( v 3) binding in endothelia l cells (200). This is of note as the integrin, v 3, is also the major integrin of ma ture osteoclasts (55, 56). Cortactin may be responsible for organization of rec eptor signaling in the region of the sealing zone as it possesses both proper spatial and temporal localization with newly forming actin networks (186, 188, 200).

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79 Cortactin may not be a direct acti vator of the Arp2/3 complex. However, the multidomain structure of cortactin, in conjuncti on with its distribution in dynamic cortical actin structures, may allow it to bridge regions of actin reorganization with receptor signaling complexes, protein tyrosine kinases, and/or to recruit proteins that may positively or negatively regulate actin polymerization (186, 188, 200). Cortactin was previously identifi ed as being preferent ially upregulated during osteoclastogenesis. In this st udy, our objective was to identify the localization of cortactin in the osteoc last and to determine its requirement for actin ring formation. Materials and Methods Western blot analysis with quantitation of cortactin Anti-cortactin antibodies were obt ained from Upstate Biotechnology (Charlottesville, VA). RAW 264.7 cells were grown as previously described, plated on 6 well plates, and either left un stimulated or stim ulated with RANKL. Cell lysates were collected from both t he control and treated ce lls. Cells were washed twice with ice cold PBS and scraped from the plates. The cells were then detergent solubilized in 0.2% Triton X-100 in PBS Equal amounts of the lysates were separated by SDS-PAGE, followed by Western Transfer. The nitrocellulose blots were then incubat ed with anti-cortactin antibodies for one hour, washed three times, incubated wit h HRP conjugated secondary antibody, washed three times, and incubated with Super Signal Dura West Chemiluminescent Substrate (Pierce, Rockford, IL). The blots were then viewed

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80 on a Fluorochem 8000 (Alpha-Innotech, San Leandro, CA), and quantitation was performed by Spot Densitometry (Fluo r-Phor Software, Alpha-Innotech, San Leandro, CA). The integrat ed density values (IDV) were obtained (white = 65535, black = 0). Background values were subtracted, and the intensities were normalized against the value of actin in the sample. The values were then compared between stimulated and unstimula ted cells. The stimulated and unstimulated values were statistically analyzed using the paired t-test, with statistical significance (p) being less than 0.05. Co-localization of cortactin with actin and Arp3 Cell culture was performed as previ ously described for RAW 264.7 cells and mouse marrow osteoclasts. To determine the co-localization of cortactin with actin in the actin ring, osteoclasts we re fixed in 2% formaldehyde, detergentpermeabilized with 0.2% Triton X-100 in PBS for 10 minutes, washed in PBS and blocked in PBS with 2% BSA (bovine serum albumin) for one hour. Actin filaments were stained with TRITC phalloid in. Cortactin was probed with an anticortactin monoclonal antibody (Upstate Biotechnology). Subunit B2 of V-ATPase was detected with an anti-B2 polyclonal antibody (34). Bound antibodies were detected by labeling with CY2 tagged anti-mouse secondary antibody. Osteoclasts were visualized using t he MRC-1024 confocal laser scanning microscope and LaserSharp software (Bio -Rad, Hercules, CA). Images were taken in sequential series to eliminate any overlap of emission and analyzed by confocal assistant software.

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81 Immunoprecipitation of actin associated proteins using a GST-cortactin construct To determine the interaction of cortac tin with actin associated proteins in osteoclasts, Glutathione S-transferase (GST)cortactin prokaryotic expression construct GST-cortactin was obtained fr om Scott Weed, Ph.D. (West Virginia University, Morgantown, WV). The GST construct was transformed into Escherichia coli strain DH5 The fusion protein was pur ified by induction of the bacterium with isopropyl-1-thio-b-D-galac topyranoside. The fusion protein was run on a glutathione-Sepharose 4B column and eluted with 10 mM reduced glutathione in lysis buffer. Cell lysa tes were obtained from RANKL stimulated RAW 264.7 cells as described previously. Prior to incubation, the cell lysates were centrifuged at high speed to remove an y actin to prevent mi sleading results. The GST-fusion protein conjugated to Seph arose was incubated with cell lysates from RANKL stimulated cells. As a c ontrol, Sepharose without the GST-cortactin fusion protein was also incubated with t he cell lysates from RANKL stimulated cells. The Sepharose was centrifuged and washed twice with binding buffer lacking ATP. Bound proteins were visua lized by Western blotting with anti-Arp3, anti-VASP, anti-E subunit of V-ATPase, anti-WASP (Sant a Cruz), and anti-actin (Sigma) antibodies after SDS-PAGE. Knocking down gene expressi on of cortactin using siRNA Five single interfering RNA (siRNA) duplexes to murine cortactin (accession no. NM_007803) were designed and produced by Sequitur (Natick, MA): 120648 (targeting bp 626-644) anti-sense 5-UCUUGUCUACACGGUC

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82 AGCTT-3, sense 5-GCUGACCGUGUAGA CAA GATT-3; 120649 (targeting bp 919-937) antisense 5GAAACCAGUCUUA UAGUCUTT, sense 5AGACUAUA AGACUGGUUUCTT-3; 120650 (target ing bp 1169-1187) antisense 5UAGCACGGAUAUUACUGGUTT-3, s ense 5-ACCAGUAAUAUCCGUGCUATT3; 120651 (targeting bp 673-691) antisense 5-AGACUCAUGCUUCUCCG UCTT-3, sense 5-GACGGAG AAGCAUGAGU CUTT -3; 120652 (targeting bp 830-848), antisense 5-UCUGCACACCAAA CUUUCCTT-3, sense 5-GGAAAG UUUGGUGUGC AGATT-3; 120653 (control) antisense 5-UGGUCAUUAUA GGCACGAUTT-3, sense 5-AUCGUGCCUAUAAUGACCATT-3. Initial experimentation showed only siRNA 120649 capable of downregulating cortactin; the other siRNAs were used as ineffective controls. In addition, a siRNA known to downregulate cortactin was obtained (Ambion part no. 60931, targeting exon 5) as well as both positive (GAPDH) and negative controls. RANKL stimulated RAW 264.7 cells on glass covers lips in 24 well plates were not transfected or transfected with either 150 nM of t he experimental or control siRNA and 2 g/ml Lipofectamine 2000 (Invitrogen) in Op ti-MEM media supplemented with RANKL on day 4 of differentiation (at the appe arance of multinucleated cells) and monitored for siRNA uptake. A fluoresc ent oligomer (part no. 2013; Sequitur) was added for uptake assessement. Six hour s after transfection, the media was replaced with DMEM with fe tal bovine serum and RANKL. The cells were incubated for 48 hours at 37oC in a CO2 incubator. They were then fixed in 2% paraformaldehyde and viewed for incorporati on of the siRNA with the use of the FITC label. Only cells labeled with FITC were identi fied as having either the

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83 control siRNA or experimental siRNA. The cells were stained with TRITC phalloidin to visualize the actin ring mo rphology. Osteoclasts were visualized using the MRC-1024 confocal laser scanning microsc ope and LaserSharp software (Bio-Rad, Hercules, CA). Images were taken in sequential series to eliminate any overlap of emission and analyz ed by confocal assistant software. To determine the downregulation of pr otein expression, RANKL stimulated RAW 264.7 cells were grown on 6 well plat es. On day 6 of differentiation, they were either not transfected or transfected wit h 150 nM of control or experimental siRNA in 10 l lipofecta mine 2000. The media was replaced with DMEM with FBS and RANKL 6 hours after transfection. The cells were incubated for 48 hours at 37oC in a CO2 incubator. The cells were scraped and washed twice with cold PBS. The lysates were centrifuged and the cell pellet was lysed on ice using 150 l cell extraction buffer (BioSource In ternational, Camarillo, CA, USA) supplemented with protease inhibito r cocktail (Sigma P2714) and phenylmethylsulfonylfluoride (P MSF) for 30 minutes, vortexing every 10 minutes. The cell lysate was then centrifuged at 13,000 rpm for 10 minutes at 4oC. Bradford assay was performed to determi ne protein concentration. Equal concentrations of proteins were separat ed by SDS-PAGE, followed by transfer to nitrocellulose. The nitroc ellulose blots were incuba ted overnight in blocking buffer, after which they were incubat ed with both anti-cortactin and anti-actin antibodies for 2 hours, followed by in cubation with secondary horseradish peroxidase labeled antibodies for 1 hour Chemiluminescent substrate was added and the blots were visualized using an Alpha Innotech Fluorochem 8000.

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84 Results Cortactin is upregulated at the transcriptional level during osteoclastogenesis Cortactin protein levels increase during osteoclastogen esis as is verified by Figure 4.2 (184). Increased expression is due to transcriptional rather than translational regulation as was identified by PCR analysi s (Figure 3.3). Unlike the other proteins tested, cortactin mRNA was not detec ted prior to treatment of RAW 264.7 cells with RANKL. Cortactin in the actin rings of resorbing osteoclasts Figure 4.3 shows represent ative micrographs of the staining of activated osteoclasts on dentine bone with anti-cortacti n and anti-Arp 3 or phalloidin. As described in previous research, in the activated osteoclast on bone slices, actin is enriched in the ring surrounding the ruffl ed membrane. Cortactin is shown to be a major element of the actin ring of resorbing osteoclasts. Cortactin is required for actin ring formation A new siRNA (120648) was identifi ed that knocked down cortactin expression (Figure 4.4). A commercial siRNA known to downregulate cortactin was also used to confirm our data (Figure 4.6). Osteoclast-like RANKL stimulated RAW 264.7 cells on 6 well plates were transfected and kept in culture for 48 H. Cortactin was not detected by Western analysis in the cells transfected with effe ctive anti-cortactin siRNAs (Figure 4.4 and 4.6).

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85 RANKL stimulated RAW 264.7 cells on gl ass coverslips were grown on 24 well plates and transfected wit h experimental or control siRNAs. The cells were incubated for 48 hours at which time they were fixed. Immunocytochemistry showed normal actin rings in the no tr eatment and control si RNA groups (Figure 4.5 and 4.7). However, a complete loss of actin ring podosomal organization occurred in the experimental group. Although there was a loss of actin rings, the cells remained viable and well spread. Cortactin-binding proteins in extr acts from osteoclast-like cells To identify actin-associat ed proteins that interact with cortactin, pull-down experiments were performed on detergent solubilized extracts of RANKL stimulated R264.7 cells. Recombinant GST-cortactin (Figure 4.8) or vehicle was added to the extracts, and then pulled do wn with Glutathione Sepharose beads, separated by SDS-PAGE and Western blotted. Consistent with previous reports, cortactin was found to interact with Arp2/ 3 complex and n-WASp (Figure 4.9). Surprisingly, we detected high levels of Vasodilator-stimulated phosphoprotein (VASP), a regulator of actin polymerization, and V-ATPase subunits (Figure 4.9). Efforts to pulldown cortactin in immunopr ecipitations of V-ATPase were not successful. However, we did identify VAS P, suggesting a potential complex that includes VASP, cortactin and, V-ATPase (Figure 3.5). Discussion As previously shown, cortactin is differentially upregulated during osteoclastogenesis (184). This preferential upregulati on in response to RANKL stimulation supports a hypot hesis that it is import ant for osteoclastic bone

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86 resorption and may be a vital component in either V-ATPase tr anslocation to the ruffled membrane or formation of the actin ring. Cortactin co-localizes with the Arp2/ 3 complex in the actin ring of osteoclasts. Previous data have shown t hat cortactin forms a tertiary complex with the Arp2/3 complex and N-WASP to activate actin polymerization and for stabilization of actin based networks (186, 188). Its identification in the actin ring supports its localization to this complex of proteins. Immunoprecipitation with the GST-cortactin fusi on protein identified associations between the Ar p2/3 complex and N-WASP, wh ich is consistent with previous studies that demonstrated the complex composed of these proteins plays a role in the regulation of actin polymerization (186, 188) Unexpectedly, cortactin also interacted with V-AT Pase and Vasodilator stimulated phosphoprotein. VASP is an ac tin associated protein that tracks the fast growing end of actin filaments (201, 202). It is still unclear as to the precise mechanism of actin; however, it may be involved in protecting growing ac tin filaments from capping proteins (201, 202). In addition, the capacity of VASP to concentrate profilactin complex near the fast growing end of actin filaments may be vital (202). This is the first report of VASP and cortactin in the sa me complex. We currently do not know whether the interact ion is direct or indirect. Potential interaction domains are present in t he two proteins. VASP contains a src homology region 3 (SH3) binding domain in the proline-rich central region (203), while cortactin has a carboxy-terminal SH 3 domain (204). Efforts are underway

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87 to determine whether these domains in teract and to explore the functional consequences of the interaction. The use of siRNA to knock down cortac tin results in a loss of actin ring formation which demonstrates that cortac tin is crucial for the formation of podosomes and actin rings in osteoclast s. Two separate siRNAs targeting cortactin greatly reduced cortactin levels and disabled the capac ity of osteoclasts to form actin rings and podosomes. Together with the fact that cortactin is specifically upregulated during osteoclast ogenesis (184), these data suggest that cortactin plays a vital role in osteoclast function. In summary, we showed that cortactin is required for the formation of the podosomes and actin rings that are vital for osteoclast function. Cortactin interacts with Arp2/3 comple x and n-WASp as expected in osteoclasts extracts (186, 188). Novel interactions betw een cortactin and VASP and cortactin and VATPase were identified. Our data are co nsistent with cortacti n playing a role in osteoclasts in the integration of cytoskeletal and membrane dynamics.

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88 Figure 4.1. Cortactin, NWASp and Arp2/3 form a synergi stic, ternary complex to initiate actin polymerization. The Arp2/3 complex is inactive in its unbound form. Activation of the Arp2/3 complex occurs through the N-WASP family of proteins binding to the Arp2 subunit. Upon activation, a confo rmation change occurs in between the Arp2 and Arp3 subunits induci ng actin polymerization. Cortactin binds to the Arp3 subunit and functions to enhance actin polymerization as well as stabilize the Arp2/3 i nduced branched actin networks. (Weaver et al. Curr Biol. 2002; 12:1270-1278) (188)

90 ACTIN CORTACTI N MERGE AR P3 CO RTACTIN MERGE Figure 4.3. Cortactin co-localizes with the podosomal core proteins, actin and the Arp2/3 complex. RA W 264.7 cells were stim ulated with RANKL to differentiate into osteoclast-like cells and fixed and stained with anti-cortactin antibody and rhodamine phalloid in or anti-Arp3 antibodies. Note that there is precise co-localization between Arp3 and co rtactin and actin and cortactin.

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91 Figure 4.4. siRNA 120649, but not a cont rol siRNA (120653), effectively knocks down the cortactin content to an undetectabl e level of osteocla st-like cell extract after 30 hours compared with actin. RAW 264.7 cells were stimulated with RANKL. Just as large, multinucleated osteoclasts began to appear, cells were transfected as noted. Cells transfected with siRNA 120649, which had proved effective at knocking down cortactin in preliminary experiments, reduced cortactin levels dramatically compared with either c ontrol cells or cells transfected with an ineffective siRNA 120653.

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92 Figure 4.5 Actin rings are di srupted in cortactin knockd own. Untransfected RAW 264.7 osteoclast-like cells or osteoclast-like cells transfected with ineffective siRNA (120653) or effective siRNA (120649) were fixed after 30 hours and examined for the presence of fluorescent oligo marker of transfection (bottom panels) or F-actin by staining with phal loidin (top panels). The photographs are representative cells. The effective siRNA disrupted the ability of the osteoclasts to form actin rings.

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93 Figure 4.6. An siRNA known to downreg ulate cortactin (Ambion) effectively knocks down the cortactin content of osteoclast-like cell extract to an undetectable level after 30 hours compared with actin. RAW 264.7 cells were stimulated with RANKL. Ju st as large, multinucl eated osteoclasts began to appear, cells were transfected as noted. Cells transfected with Ambion siRNA, which is known to knock down cortactin levels, reduced cortactin levels dramatically compared with ei ther control cells or cells transfected with either positive or negative controls.

95 Total Purified Protein GST-Cortactin Extract Figure 4.8. Transformation and Purificati on of GST-cortactin fusion protein. A GST-cortactin fusion protei n was obtained from Dr. Scott Weed (West Virginia University, Morgantown, WV). The constr uct was transformed into E.coli strain DH5a and induced with IPTG. The fusi on protein extract was run on a glutathione-sepharose column and el uted with reduced glutathione. Cortactin

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96 G-S/C/L G-S/ L IP: GST-Cortactin Probe: Cortactin Arp3 VASP N-WASP E Subunit of V-ATPase Actin Figure 4.9. Immunoprecipitation Ex periments with GST-Cortactin Show a Linkage between Cortactin and Arp3, VAS P, N-WASp and the E Subunit of VATPase. Cells lysates from RANKL stimulat ed RAW 264.7 cells were incubated with glutathione sepharose with or with out GST-cortactin. The lysates were washed and eluted in loading buffer. They were separated by SDS-PAGE and Western transfer. Bound proteins were th en visualized by pr obing with anti-Arp3, anti-cortactin, anti-VASP, anti-N-WASP, anti-E s ubunit, and anti-actin.

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97 CHAPTER 5 THE ROLE OF VASP IN OSTEOCLASTOGENESIS Introduction Like cortactin, numerous additional proteins have been identified as components of the cytoskeletal machinery. VASP is one such protein. It may act directly as a nucleator of the Arp2/3 comp lex or indirectly as a structural scaffold for signaling and cytoskeletal proteins such as vinculin, ActA, zyxin, and Fyb/Slap (149, 151). VASP is a 46 kD protein (203, 205) originally is olated from human platelets and is the foundi ng member of the Ena/VAS P family composed of Vasodilator-Stimulated Phosphoprotein (VASP), mammalian Enabled (Mena), and ENA/VASP-like protein (Evl ) (Figure 5.1) (203, 205, 206). This family of proteins plays a key role in actin bas ed motility and is localized predominantly at focal adhesions, cell/cell contacts, and regions of highly dynamic actin reorganizations such as lamellipodia (151, 203). The VASP protein contains three primary domains, EVH (Ena/VASP Ho mology domain) I, proline rich, and EVH2 (189, 203, 206). The EVHI domain is located at the N-terminus and binds actin related proteins such as zyxin vinculin, and ActA (189, 203, 206). The proline rich region interacts wit h proteins containing SH3 and WW domains and contains a 4 GP5 motif which is the binding site of profilin, a G-actin

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98 regulatory protein (189, 203, 206). The EVH2 domain contains the actin binding site and is the location for oligom erization (189, 203, 206). VASP is phosphorylated by both the Protein Kinase A (PKA) and Protein Kinase G (PKG) pathways (207). The phosphorylated protein has an apparent weight of 50 kD (205, 207) PKA preferentially phos phorylates VASP at Ser157 which is located N-terminal to the (GP5)4 profilin binding site in the proline rich region (189, 203, 206). This phosphorylati on site is in close proximity to the ligand binding module which in turn alters the ligand binding properties (189, 203, 206). In addition, it al so phosphorylates Thr274 although the consequences of this phosphorylation ar e not fully understood (189, 203, 206). The PKG pathway preferentially phosphorylat es Ser239, but like PKA, will also phosphorylate Thr274 (189, 203, 206). Phosphorylation by the PKA pathway has been shown to diminish F-actin binding, suppressing actin nucleation as well as inhibiting Arp2/3 triggered actin polymeriz ation; thus, it can be a negative regulator of actin polymerization (189, 203, 206). The PKA pathway is activated in mu rine osteoclasts in response to calcitonin (207). Calcitonin is a known i nhibitor of bone resorption and is used to treat metabolic bone diseases such as osteoporosis and Paget's disease (190, 207). The calcitonin receptor, a 7 trans membrane G-protein coupled receptor, is located on the cell surface of osteoclast s (191, 192). Activation by calcitonin signals the receptor to activate the PKA pathway (190, 191). This could lead to phosphorylation of the VASP protein and in turn to the changes in the organization of F-actin that are known to occur in response to calcitonin.

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99 In this study, our objective was to examine the role of VASP in osteoclastogenesis. We sought to det ermine the localization of VASP in the osteoclast and well as its requirement fo r actin ring formation. In addition, we sought to determine what effect phosphor ylation of VASP would have on the actin ring of osteoclasts. Materials and Methods Distribution of VASP in the actin ring Cell culture was performed as previous ly described. For identification of VASP localization, the cells were fixed in 2% formaldehy de, solubilized in 0.2% Triton X-100 in PB S and blocked in PBS with 2% BSA. Cells were stained with rhodamine phalloidin or antibodies recogni zing VASP at a dilution of 1:100 in PBS. Secondary antibodies were dilu ted according to manufacturers instructions. Osteoclasts were visual ized using the MRC-1024 confocal laser scanning microscope and LaserSharp softwar e (Bio-Rad, Hercules, CA). Images were taken in sequential series to e liminate any overlap of emission and analyzed by confocal assistant software. Effects of calcit onin on actin rings of osteoclasts Cell culture was performed as previously described. On day 6 of differentiation (many large multinucleat ed cells present), calcitonin (10nM) was added to the cells and incubated for time poi nts of 1, 2, and 24 hours. For identification of morp hological characteristics, the cells were then fixed in 2% formaldehyde, solubilized in 0.2% Trit on X-100 in PBS and blocked in PBS with 2% BSA. Cells were stai ned with rhodamine phalloidin or antibodies recognizing

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100 Arp 3 or phospho-VASP (Ser 157) at a dilution of 1:100 in PBS. Secondary antibodies were diluted according to manu facturers instructions. Osteoclasts were visualized using the MRC-1024 co nfocal laser scanning microscope and LaserSharp software (Bio-Rad, Hercules, CA ). Images were taken in sequential series to eliminate any overlap of emi ssion and analyzed by confocal assistant software. For assessment of protein expression, RANKL stimulated RAW 264.7 cells on 6 well plates were either untr eated or treated with calc itonin (10 nM) for 1, 2 or 24 hours. Cells were then scraped and washed twice with PBS. The pellets were lysed using 250 l of cell extraction buffer (B ioSource International, Camarillo, CA, USA) supplemented with protease inhibitor cocktail (Sigma P2714) and phenylmethylsulfonyl fluoride (PMSF) for 30 minutes on ice with vortexing every 10 minutes The extract was centrifuged for 10 minutes at 13,000 rpm at 4o C. Bradford assay was perfo rmed on the lysates. Equal concentrations of protein were separat ed by SDS-PAGE, followed by western transfer. The nitrocellulose blots were blocked in blocking buffer overnight and incubated with both antiVASP and anti-phospho-VASP (S er 157) antibodies for 2 hours. The bots were washed and incubated with a horseradish peroxidase (HRP)-labeled secondary antibody for 1 hour, followed by incubation with a chemiluminescent substrate. The blots we re visualized usin g an Alpha Innotech Fluorochem 8000. Quantitation wa s performed using densitometric measurements of integrated density values.

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101 VASP-null colony To determine the effects of knocking out VASP expression in osteoclasts, three female heterozygous mice and one homozygous VASP knockout male mouse were obtained as a generous gift fr om Dr. Ulrich Walter (Institute of Clinical Biochemistry and Pathobiochemistr y, Wurzberg, Germany). A breeding colony was initiated with appr oval from the University of Florida Institutional Animal Care and Usage Committee. Ba sed on Mendelian genetics, half of each litter should be homozygous knock out mi ce and half should be heterozygous. After weaning, a tail sample from eac h pup was obtained and RNA was extracted with RNAeasy Mini Kit (Qiagen, Valenc ia, CA) following the manufacturers instructions and quantified by spectr ophotometer. The sequence for VASP was obtained from Gen Bank. Primers were designed as follows: forward 5GAGGAGCTGGAACAACA GAA-3; reve rse 5-CCAGGCAGGAAGTACA GAAA3. For standard RT-PCR, 3 ug of total RNA were anneale d to an oligo-dt primer and first strand cDNA synthesis was performed using Thermoscript RT-PCR System (Invitrogen, Carlsbad, CA) follo wing manufacturers directions. Onetwentieth of the cDNA was subjected to amplification by PCR. PCR was performed under the follo wing conditions: 95oC for 2 minutes, then 35 cycles of 90oC, 30 seconds; 58oC, 30 seconds; 72oC, 30 seconds. O ne-half of the PCR product was separated on 0.5% agarose gel with ethidium bromide staining for 1 hour. Images were detected using UV tran sillumination on a Fluorochem 8000 (Alpha-Innotech, San Leandro, CA). Homozygous mice were determined to be those by which PCR with multip le primers was unsuccessful.

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102 Mouse marrow osteoclasts were grown from marrow derived from the long bones of the hind legs of the homozy gous VASP knockout and the heterozygous mice. The marrow cells were grown in -MEM medium with 10% fetal bovine serum (FBS) plus 10-8 M 1,25-dihydroxyvitamin D3 for a period of approximately seven days. The cells were then scraped, plated on 24 well plates, and treated with calcitonin (10nM) for 1 hour. The ce lls were fixed in 2% paraformaldehyde, detergent-permeabilized with 0.2% Triton X-100 in PBS for 10 minutes, washed in PBS and blocked in PBS with 2% BSA (bovine serum albumin) for one hour. Actin filaments were stained with TRITC phalloidin. VASP was probed with an anti-VASP polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Bound antibodies were det ected by labeling with CY2 tagged anti-rabbit secondary antibody. Osteoclasts were visualized using t he MRC-1024 confocal laser scanning microscope and LaserSharp software (Bio-Rad, Hercules, CA). Images were taken in sequentia l series to eliminate any overlap of emission and analyzed by confocal assistant software. This protocol was approved by the University of Florida Institutional Animal Care and Usage Committee. PCR analysis of the ENA/VASP family member, Evl RNA was extracted from RANKL different iated RAW 264.7 cells as well as from unstimulated RAW 264.7 cells using RNAeasy Mini Kit (Qiagen, Valencia, CA) and quantified by spec trophotometer. The sequ ence for evl was obtained from Gen Bank. The following pr imers were designed: forward 5ACCAGCAGGTTGTGATCAAT-3; revers e 5-AATAGACCCGGTGTTCT GTG-3. For standard RT-PCR, 3 g of total RNA were annealed to an oligo-dt primer and

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103 first strand cDNA synthesis was perfo rmed using Thermoscript RT-PCR system following manufacturers directions. One-tw entieth of the cDNA was subjected to amplification by PC R using the above mentioned primers. PCR was performed under the following conditions: 95oC for 2 minutes, then 35 cycles of 90o C, 30 seconds; 58oC, 30 seconds; 72oC, 30 seconds. One hal f of the PCR product was separated on 0.5% agarose gel with et hidium bromide staining for 1 hour. Images were detected using UV trans illumination on a Fluorochem 8000. Results VASP is present in the actin rings of osteoclasts. The actin rings of osteoclasts were stained with anti-VAS P or phalloidin. Figure 5.2 is a representat ive micrograph of the staini ng. Co-localization was observed between VASP and actin. VASP is phosphorylated at Serine 157 in response to calcitonin treatment and results in the disruption of the actin ring of osteoclasts. Osteoclasts were treated with calcitonin at baseline, and cells were fixed and stained with phos pho-VASP Serine 157 or Arp3 ant ibodies at 1, 2 and 24 hour time periods (Figure 5.3). Osteoclasts at baseline showed no phosphorylation of VASP. Treat ment with calcitonin ca used a phosphorylation of VASP at Serine 153 as obser ved by the increased signal intensity at 1 and 2 hours. This phosphorylation coincided wit h a disruption of the microfilament organization in the actin rings from tight ly focused rings to broad bands of actin. By 24 hours, the phosphorylat ion and actin ring morphology were returning to baseline levels.

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104 Western analysis of the ca lcitonin-treated osteoclast s showed a three fold increase in phosphorylation of VASP at Se rine 157 at 1 and 2 hour time points (Figure 5.4). This confirms VASP is pho sphorylated in response to calcitonin treatment. The osteoclasts of mice lacking the VASP gene are able to form actin rings. Heterozygous female and homozygous male knockout mice were obtained from Ulrich Walter (Institute fo r Clinical Biochemistry and Pathobiology, Medizinische Universittsklinik, Wrzburg, Germany). The mi ce were bred and homozygous VASP-null mice were identified by tail DNA isolat ion (Figure 5.5). Osteoclasts were cultured from the mous e marrow of the hind legs of the VASP deficient mice. The osteoclasts were then fixed and stained with phalloidin or treated with calcitonin and fixed and stained with phalloidin. Normal actin ring morphology was observed in osteoclasts from VASP-null mice (Figure 5.6). Treat ment with calcitonin, which disrupts actin ring morphology by the PKA pathway disrupted the actin rings of both the control, as expected, and the VASP null osteoclasts. This suggests that calcitonin may exert its functions through anot her VASP/Ena family member. Evl is upregulated in response to osteoclast differentiation To identify other members of the ENA/ VASP family that could play a role in osteoclastogenesis, PCR was performed using primers to detect changes in gene expression in evl. Unlike VASP, Ev l mRNA was preferentially upregulated during osteoclastogenesis, with a complete la ck of detection prior to treatment of RAW 264.7 cells with RANK-L (Figure 5.7).

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105 Discussion Vasodilator stimulated phosphoprote in is a member of the ENA/VASP family of proteins (190, 192, 203, 206, 207). These proteins localize to areas of dynamic actin polymerization and are know n downstream effectors of multiple signaling pathways (189). These studies show that VASP is a component of the actin ring of osteoclasts, which is cons istent with its localization to areas of dynamic actin reorganization (205). The function of VASP in the actin ring of osteoclasts is still unknown. However, ENA/VASP proteins bind directly to Gactin and F-actin as well as profilacti n and are known to promote the elongation of actin filaments by recrui ting profilactin complexes to the sites of dynamic actin reorganization (205, 208-210). VASP also functions to enhance Arp2/3 activity and prevent capping proteins from inhibi ting actin polymerization (210). These data strongly suggest that VASP plays a key role in actin dynamics. VASP has been identified as a subs trate for both the PKA and PKG phosphorylation (208, 211, 212). Platelet s from VASP null mice have defective PKA signaling and exhibit deficiencies in platelet aggregation (213-215). Calcitonin is a known activator of the PKA pathway and induces changes in the cytoskeleton (191, 192). Treatment of osteoclast s with calcitonin shows a three-fold increase in phos phorylation of VASP at Seri ne 157 within the first two hours of treatment with a return to base line by 24 hours. The actin rings of calcitonin-treated osteocla sts were disrupted as the microfilament organization changed from a tightly focused ring to bro ad bands of actin. By 24 hours, the actin ring morphology had re turned to baseline morphology This is consistent

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106 with data indicating that VASP plays a ro le in actin filam ent organization by affecting the branching activity of the Arp2/3 complex. Upon activation of VASP, the density of Arp2/3 induced branching is decreased, resulting in larger and more sparsely branched filaments (216) Upon deactivati on, Arp2/3 mediated actin polymerization and branching occurs resulting in a dense, tightly branched network. Although data confirm that VASP is a ssociated with the reorganization that occurs in the actin ring of osteoclast s, VASP null mice hav e no real skeletal deficiencies (212-214). Ost eoclasts from VASP null mi ce exhibit normal actin ring morphology. In addition, when treated with calcitonin, the osteoclasts exhibit actin ring disruption similar to that obser ved in control cells. These findings support the lack of skeletal deficiencies id entified in VASP null mice and suggest that VASP may not play a major role in th e dynamic actin polymerization found in the podosomes of the actin ring. The ENA/VASP family consists of three mammalian members, VASP, Mena (mammalian Enabled) and Evl (Ena/ VASP-like protein) (211, 217). PKA phosphorylation, as induced by calcitonin, is known to affect two members of the ENA/VASP family, VASP, as our data have shown, and Evl (203, 211). Evl is highly expressed in cells of hema topoietic lineage and has been shown to nucleate actin polymerization (203, 206). In addition, phosphorylation of Evl results in a decrease in nucleation activi ty (211). These data suggest that Evl may be the Ena/VASP family member that plays a key role in osteoclastogenesis. PCR analysis of unstimulated and RANKL stimulated RAW

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107 264.7 cells indicates that Evl is prefer entially upregulated in response to RANKL stimulation, as is seen with cortactin. This pref erential upregulation suggests it functions in the dynamic actin reorganiza tions that occur during osteoclastic differentiation. In summary, VASP is present in the actin rings of osteoclasts and is phosphorylated at Serine 157 in response to calcitonin treatment, which activates the PI3K pathway. This activation caus es a disruption of the actin ring of osteoclasts. Although treat ment with calcitonin may indicate a role for VASP in actin ring formation and maintenance, we did not detect any skeletal defects in the VASP knockout mouse. Osteocla sts cultured from VASP knockout mice respond similarly to those from control mi ce, indicating another ENA/VASP family member may play a more dominant role in osteoclastogenesis. Evl, an ENA/VASP family member, is present in cells of hematopoietic lineage (14) and has been shown to be preferentially upregulated in response to RANKL treatment. Evl may be responsible for t he structural changes se en in actin ring morphology when treated with calcitonin.

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108 Figure 5.1. The E na/VASP family. Cartoons of t he three mammalian members of the Ena/VASP family are depicted. All three members share a similar domain structure which consists of an aminoterminal EVH1 domain, a central prolinerich region, and a carboxy-terminal EVH2 domain. In addition, all mammalian Ena/VASP proteins share an amino-termi nal PKA/PKG phosphorylation site (Ser157 of VASP). (Kwiatkowski AV et al. Trends Cell Biol. 2003; 13(7):386-92) (206)

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109 Actin VASP Figure 5.2. VASP is present in the actin ring of osteoclasts. Mouse marrow osteoclasts were cultured on bovine dentin slices fo r 2 days and then fixed and stained with rhodamine phalloidin and anti-V ASP antibody. VASP is observed to co-localize with actin in the podosomes of the actin ring.

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110 B aseline 1 hour 2 hours 24 hours PhosphoVASP Arp3 Figure 5.3. VASP is phosphorylated at Se rine 157 in response to calcitonin treatment and results in the disruption of the actin ring. RAW 264.7 cells were cultured with RANKL until mu ltinucleated osteoclast-lik e cells were observed. The cells were then treated with 10 nM calcit onin and fixed at bas eline, 1, 2, and 24 hour time points. The cells were st ained with antibodies re cognizing Arp3 and phospho-VASP Ser 157. Calcitonin trea tment caused a phosphorylation of VASP at 1 and 2 hour time points but returned to base line by 24 hours. A broadening of the actin ring coincided with the observed phosphorylation.

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111 Figure 5.4. Calcitonin induc es a three fold increase in phosphorylation levels of VASP at Serine 157. Cell lysates were collected from the RAW 264.7 cells treated with calcitonin at bas eline, 1 hour and 2 hours. A Bradford assay was performed to standardize prot ein concentrations. The lysates were separated by SDS-PAGE and western analysis and pro bed with either antiPhospho-VASP (Serine 157) or anti-VASP antibodies. Quantitation was performed on the western blots by densitomet ry measuring integrated dens ity values.

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112 Figure 5.5. Identificati on of VASP null mice from breeding of heterozygous female with a homozygous male. RNA was extracted from the tail of each pup in the breeding colony. Prim ers were synthesized again st VASP to determine which mice were lacking the VASP gene. The white circle ident ifies the presence of the VASP gene, while the black circle identifies a VASP nu ll mouse. These identified VASP null mice were then used for immunocytochemical studies.

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113 Figure 5.6. Osteoclasts of mice la cking the VASP gene are able to form actin rings and respond to calcitonin in the same fashion as control cells. Osteoclasts were cultured from the m ouse marrow of the hind legs of VASP null or control mice. The osteoclasts were either untr eated or calcitonin ( 10 nM) treated for 10 minutes and then fixed and stained with rhodamine phal loidin. The VASP null osteoclasts form actin rings like the controls. In addition, treatment with calcitonin, which disrupts actin ring morphol ogy, similarly disrupts the actin ring in both control and VASP null osteoclasts.

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114 Unstimulat ed Stimulated EVL GAPDH Figure 5.7. Evl, a mem ber of the ENA/VA SP family, is upregulated in response to osteoclastogenesis. RNA was extracted from RAW 264.7 cells unstimulated or stimulated with RANKL. Primers were designed again st Evl, a member of the ENA/VASP family. RT-PCR identifies preferential upregulation of Evl in response to RANKL stimulation.

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115 CHAPTER 6 MODEL AND FUTURE DIRECTIONS The Model and Hypothesis This project has tested two novel hy potheses regarding t he actin ring of osteoclasts and the association of actin ring proteins with V-ATPase. Our model first proposes that upon activation of t he osteoclast, the cell becomes polarized, and the Arp2/3 complex is recruited to t he apical membrane. It is hypothesized that the actin polymerizati on that ensues is Arp2/3 m ediated and forms the actin ring. It is possible that this polym erization produces force at the plasma membrane, driving the membrane into the bone and forcing it to conform, creating a tight, yet dynamic seal. To c ounter the force being applied to the bone at the sealing zone, int egrin-mediated focal adhesions elsewhere on the apical surface maintain the osteoclast in posit ion. This hypothesis would account for the dynamic nature of the actin ring and sealing zone as well as the specific exclusion of integrins from t he area of the sealing zone. In addition, it is also hypothesiz ed that the Arp2/3 complex or its associated proteins may bind V-ATPa se and that Arp2/3 mediated actin polymerization may be involved in translocation of V-ATPase to and from the ruffled membrane. The association of V-ATPase with actin based networks is first confirmed by the actin binding abilit y of V-ATPase (9, 11, 218). Several V-

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116 ATPase subunits have been identified to bind actin (9, 11, 218). In addition, in inactivated osteoclasts, F-actin and V-ATPa se co-localize in cytosolic vesicles (9). This co-localization is only di srupted upon activation of the cell, where VATPase is then inserted into the ruffled membrane and actin is localized to the area of the sealing zone (9). Due to the highly dynamic nature of Arp2/3 mediated actin polymerization and the clos e proximity of the ruffled membrane and actin ring, this would seem a plausible mechanism. This study has identified many important characterist ics of the actin ring of osteoclasts. We have shown for th e first time that the dynamic actin polymerization in the actin ring of osteoclasts is Arp 2/3 mediated. Osteoclast actin rings are now identified as being composed of discrete and dynamic actin based structures, termed podosomes (33, 34, 43). Presented immunocytochemical data confirms the cu rrent literature t hat podosomes are composed of a core of actin and asso ciated proteins, such as the Arp2/3 complex, cortactin and VASP (35, 36). These proteins, when viewed in zsection, are concentrated at the resorptive surface, which is consistent with their role in the force production at the reso rptive surface and their incorporation of actin into filaments at the resorpti ve surface and treadmilling toward the basolateral membrane. In addition, focal adhesion proteins, such as vinculin, do not co-localize with the acti n ring but surround the actin ring, as a cloud (33, 34). Subsequent to our findings, Jurdic et al. (43) redefined the parameters of the podosomes of the actin ring. The ac tin ring podosomes differ from individual

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117 podosomes in that the core proteins surround the entire ri ng instead of each individual podosome, as we observed with vinculin (43). The dynamic nature of these podosomes was confirmed by various studies. First, rhodamine-actin incorpor ated into the actin ring of saponinpermeabilized RANKL stimulated RAW 264. 7 cells within 10 minutes after treatment. In addition, the inclusion of latrunculin A, which sequesters G-actin, inhibits loss of podosomal structure. Th is indicates that new polymerization is inhibited while original filaments are treadmilling and disassembling. Second, treatment with various chem ical agents known to disrupt actin ring structures, such as wortmannin, calcit onin and cytochalasin D, also show rapid dissolution and relocation of the podosomes in osteoclasts. Upon stimulation by various factors, proteins are ofte n upregulated in response to specific functions in cells. For the osteoclast, st imulation by RANKL and CSF cause the osteoclast to polarize and specialized struct ures specific to the resorbing osteoclast to form, specif ically the ruffled membrane and the actin ring (8). We identified by PCR and wester n blot analysis two proteins that were upregulated in response to RANKL stimul ation. The upregula tion of Arp2 and Arp3 at a translational level and cortactin at a transcrip tional level suggest that these proteins play specia lized roles in osteoclastog enesis. In addition, their known association with actin related comple xes suggests that this role is in the formation of the actin ring. The function of the Arp2 and cortacti n proteins on actin ring formation and osteoclast function were examined via k nock down by siRNA. Knock down of

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118 either protein resulted in a decreas e in actin rings, confirming actin polymerization is mediated by the Arp2/3 complex. It was interesting to note that the Arp2 knock down cells appeared apoptotic while the cortactin knock down cells appeared viable. These data may sup port the role of the Arp2/3 complex in cell viability as well as its resorption function although a loss of actin ring formation cannot directly confirm a loss of bone resorption. Chellaiah et al. (165) showed initially that the gelsolin knock out mouse, which appeared to lack a podosomal-based actin ring, was capabl e of bone resorption albeit much reduced. The hypothesis is that alter native mechanisms of adhesion, such as integrins, are capable of maintaining adequate ad hesion for the resorption compartment to remain intact. 3 -/mice show decreased bone resorption, abnormal ruffled membranes, and increased osteoclast number, most likely caused from stimulation by hyperparathyroidism secondary to the hypocalcemia produced by decreased bone resorption (52). Skeletal remodeling in the 3 -/mice proceeds even in the absence of v 3; it is hypothesized that an adequate resorption rate is achieved by the increas ed number of osteoclasts, even in the presence of decreased resorption per osteoc last (52). Furt her studies with the gelsolin knockout mouse identify a W ASP-containing actin ring, capable of maintaining bone resorption, with sli ghtly reduced efficiency (219). These studies suggest that actin ring formation, in addition to integrins, is important for osteoclastic bone resorption as studies have shown that a lo ss of actin ring formation is concurrent with a reduction in bone resorption (31). The fact that reduced bone resorption has been identified ev en in the absence of the actin

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119 rings suggests that the osteoclast has alternative adhesive mechanisms to support an extracellular resorption compar tment (219). It is reasonable to postulate that for efficient bone re sorption, both integr in-based adhesion and actin ring formation are necessary. Arp2/3 mediated actin pol ymerization is known to proceed via a complex of proteins, including the Arp2/3 comple x, n-WASP, and cortactin (135, 188). The Arp2/3 complex is inactive in its unbound form (136, 137). Activation of the Arp2/3 complex occurs via its interaction with members of the N-WASP family of proteins (136, 137). This interaction causes a conformational change in the structure of the complex, inducing acti n polymerization (136, 137). Based on the data generated, as loss of Arp2 and cortacti n disrupted actin ring formation, it was logical to propose that actin ring poly merization was a function of this ternary complex. Immunoprecipitat ion experiments with a GSTcortactin fusion protein pulled down Arp3, N-WASP, VASP, and t he E subunit of V-ATPase. Although we cannot confirm if the bindi ng is direct or indirect, th e pull down of Arp3 and NWASP is consistent with the formation of this ternary complex. The binding of VATPase and VASP were unexpec ted. The ability of cortactin to pull down the E subunit of V-ATPase may ident ify an additional binding par tner for V-ATPase that may be involved in its translocation to and from the membrane. Taken together, these data support t he initial hypothesis generated for this project that actin ring formation is a result of Arp2/3 mediated actin polymerization. Based on new methods rec ently presented, further insights into

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120 the effects of a loss of actin ring forma tion on bone resorption will be able to be elucidated (Future Directions). The hypothesis that V-ATPase is translocated throughout the cell via Arp2/3 mediated actin polymeriz ation is attractive as it poses a highly dynamic mechanism of movement and responds appropriately to several chemical mediators. However, curr ent research does not suppor t this hypothesis as we have been unable to show a direct lin k between V-ATPase and the Arp2/3 complex. A limitation of this study was that only t he actin binding sequence of the B1 subunit was used to identify an interaction with th e purified Arp2/3 complex. This sequence was used si nce the Arp2/3 comple x and actin share extensive sequence homology; it would seem logical that they may also interact via the same binding site. Unfortunately, interaction was not detected via this sequence. Although a link was not di rectly identified between the Arp2/3 complex and V-ATPase, immunoprecipitatio n experiments with the B-subunit of V-ATPase and signal transduction array did identify VASP as a potential binding partner to VATPase. VASP is known to be phosphorylated in response to activation by the PKA and PKG pathways (203, 207). Previous data also show that calcitonin, which activates the PKA pathway, disrupt s actin rings (190, 191). When taken together, these data suggest that actin ring disruption may result from the phosphorylation of VASP. VASP is an acti n-associated protei n that tracks the fast growing end of actin filaments (201). The prec ise role of VASP remains unclear. However, it may be involved in protecting growing actin filaments ends

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121 from capping proteins (201). Data have al so been presented that indicate that VASP has the capacity to conc entrate profilactin comple x near the fast growing end of actin filaments and this may be vi tal to ensuring the rapid treadmilling of podosomes (202). One caveat to the ro le of VASP in osteoclastic bone resorption is that VASP knock out mice have no detected skeletal defects (213, 214). This study identifies that, in fact VASP is present in the actin rings of osteoclasts and is phosphorylated in respon se to calcitonin treatment. However, based on immunocytochemical experiment ation of osteoclasts of VASP knock out mice, there is no effect on actin ring formation. In addition, the response of the VASP knock out osteoclasts to calcitonin is equal to that of the control. These data suggests that VASP may not play a vital role in osteoclastic bone resorption. However, the Ena/VASP fam ily member, EVL, identified specifically in cells of hematopoietic lineage (211), wa s found in this study to be preferentially upregulated in response to osteoclastoge nesis. This protein warrants further study (Future Directions). Future Directions There are several future directions for this research. Previous siRNA knock down studies have lacked the ability to clearly define effects on in vitro bone resorption due to an extremely low e fficiency of transfection of mouse marrow cultures. Although RAW 264.7 cells can be efficiently transfected, VATPase is not properly located to t he ruffled membrane and thus bone resorption does not occur adequately for experiment ation. Although the mechanisms of actin ring formation can be studied as there seems to be no effect on the actin

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122 ring, bone resorption assays in RAW cells are suspect. Rece nt literature has identified a novel method of transfection of siRNAs into mouse marrow cultures (220). Experimental siRNA, along with RNAse inhibitors, are added directly to the mouse marrow osteoclasts prior to scr aping and transferring to bovine dentin slices. The siRNA is taken up during tran sfer onto bone slices. This technique was recently published by Hu et al. ( 220), who showed successful knock down of the a3 subunit of V-ATPase by this met hod. Subsequent to th at study, we also tested a known siRNA against the a3 su bunit of V-ATPase on mouse marrow osteoclasts. Protein ex pression was reduced approx imately 70-80%, which is extremely efficient for mouse marrow osteoc lasts. If we are able to get this efficiency when knocking down Arp2 or cort actin with siRNA, we will be able to examine the effects on bone re sorption in vitro. Our hypothesis also focuses on the ac tin ring being responsible for force production required for the formation of an external resorption compartment. Arp2/3 mediated actin polymeriz ation is known to be c apable for force generation as is seen in the actin-based mot ility of certain pathogens such as Listeria, Shigella and Rickettsia and the enveloped virus vacci nia (151-153). This actin polymerization that serves as the basis fo r this movement results in an actin comet tail. This movement is involved in the spread of the pathogens from cell to cell (149, 150). Based on the capabilit y of Arp2/3 mediated force generation, the data supporting the requirem ent of the actin ring fo r efficient bone resorption and the extremely tight adhesion at the region of the sea ling zone, it is plausible that this adhesion is produced by force gener ation. A long term goal is to study

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123 force generation of the actin ring of osteoc lasts using deformable membranes. If force was generated against a deformable memb rane, it should be identified as a divot in the membrane. Another goal is to identify a link between Arp2/3 mediated actin polymerization and V-ATPase translocation. Recent st udies have shown that VATPase directly binds to aldolase, whic h functions in glycolysis (26, 27). In addition, aldolase has been shown to inte ract with actin and actin-associated proteins such as WASP and cortactin ( 221, 222). These data suggest that aldolase may function as a link between the Arp2/3, cortactin, N-WASP ternary complex and V-ATPase. The dynamic inte ractions that interplay between these components may prove to be vital for trans location of V-ATPase to and from the plasma membrane. The identification of di rect or indirect interactions between these proteins can be ident ified via immunoprecipitatio n experiments using GSTcortactin or GST-VCA domai n of N-WASP constructs (Dr. Scott Weed, West Virginia University Morgantown, WV) or sp ecific antibodies. Once direct binding partners are identified, specific binding sequences will be determined, and identification of the func tional consequences examined by constructing fusion proteins with mutations in t he binding region. The identification of another ENA/VASP family member preferentially upregulated during osteoclastogenesis merits further identification. Based on literature studies, evl, whic h is highly expressed in hem atopoietic cells (211), is the least studied of the three family member s. Evl may function as the regulator of profilactin at the active sites of acti n polymerization, which, if altered, may

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124 affect actin nucleation rates. Characteri zation of this protein in the osteoclast model may give many valuable insights into actin ring formation. Initial studies will focus on the localization of evl usi ng immunocytochemical experiments. If co-localized with the actin ring, further st udies on the requirement of evl for actin ring formation using knock down studies wi ll be performed. The loss of evl may not completely disrupt actin ring forma tion but may slow down polymerization; actin polymerization assays might prove usef ul to identify rate changes based on the presence or absence of evl. Significance of Study Bone homeostasis is the maintenance of a delicate balance between two opposite and dynamic processes, bone formation and resorption (2). Bone resorption is a mandatory event in the no rmal physiological functioning of the human body, required for such processe s as human growth, tooth movement, and the maintenance of plasma calcium levels (223). Bone resorption also has extensive implications in disease proc esses. Enhanced bone resorption is associated with diseases such as malignant hypercalcemia, osteoporosis, osteolytic dysplasia, and metastatic bone tumors (224, 225). Such osteolytic lesions mediate bone resorption by either increasing osteocla stic stimulatory factors to activate differentiation of pr ogenitor cells, activating mature osteoclasts directly, or inducing the i mmune system to release additi onal factors to stimulate bone resorption (225, 226). Osteoclastic ce lls observed in disease states are in general larger in size and number, resulting in an increased resorptive activity and efficiency (227). Morbidity associated with such resorptive diseases includes

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125 pain, pathological fractures, debilitati on, and deformity (225, 226). Although these diseases all have diffe rent etiologies, in each disease, a resorption lacunae must be segregated by the sealing zone creating the acidic extracellular compartment, by which the mineralized bone is resorbed (54, 226). This research has focused on identif ying the proteins functioning in the formation of the sealing zone. We have iden tified for the first time the presence of Arp2/3 in the actin ring and that acti n polymerization proceeds via an Arp2/3 mediated process as is shown by knock dow n of the protein. This process, in contrast to those utilizing formins or SPIRE (228, 229), results in networks of densely branched actin filaments, consist ent with podosome stru cture (33). We have also identified that these branched networks are very dynamic and fairly sensitive to the surrounding environment. At this time, we have not established a mechanism for Arp 2/3 mediated V-ATPa se translocation. However, based on these data, it is proposed that the Arp2/3 complex can pose as a target to alter osteoclast function. We have also identified cortactin as an important protein in the actin ring of osteoclasts. Cortactin stabilizes the Arp2/3 mediated branched filaments (185). The knock down of cortactin in th e actin ring of osteoclasts results in a loss of podosomal arrangement of the actin ring but the ce lls remain viable. This finding is significant as cortactin has al so been implicated in cancer metastasis through the formation of podosomal-like invadopodia (230). The modulation of this protein may allow for alterations in bone resorption and cancer metastasis.

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126 Although VASP initia lly appeared very promisi ng in both its effects on actin ring function and translocation of V-ATPase to and from the ruffled membrane, experimentation proved other wise. VASP is phosphorylated in response to the protein kinase A pat hway (203, 207), which can cause a loosening of the actin ring. However, the osteoclasts from VASP knock out mice had normal actin ring morphology and no ske letal deformities were detected (213, 214). The benefit of studying VASP is the identif ication of another ENA/VASP family member, ev l. Evl may prove to be t he family member involved in actin ring dynamics. The clinical significance of this pr oject is the ident ification of the podosomal actin ring proteins and the effects of their knock down. It is known that podosomes are not only involved in physiological processes but also in disease states such as cancer (231). We have identified that the knock down of cortactin and Arp2 causes a disruption of podosomal organization. Based on our findings, it is hypothes ized that these proteins could be targeted using viral vector therapy and/or osteocla st specific promoters to re gulate osteoclastic bone resorption or possibly inhibit cancer metastasis.

148 BIOGRAPHICAL SKETCH Irene Rita Maragos Hurst is the daughter of Drs. Nicolas and Thelma Maragos, and was born and raised in t he St. Petersburg area. She was educated at Keswick Christian School and wa s president and vale dictorian of the Class of 1991. Dr. Hurst attended the University of South Florida (1991-1996) where she received degrees in both biol ogy and science education. During her time at USF, Dr. Hurst was actively involved in numerous honor and social organizations, including Kappa Delta Soro rity. In 1997, she took a leave from college to teach 7th grade math and science at McLane Middle School in Brandon, FL. The following year, Dr. Hurst enrolled at the University of Louisville College of Dentistry where s he received her Doctor of Dental Medicine degree as well as her Master of Science in oral biology. In 2001, Dr. Hurst began a joint Ph.D./residency program at the Universi ty of Florida. She completed her orthodontic training in 2006 at the University of Flori da College of Dentistry as well as her Ph.D. in biomedical scienc es through the College of Medicine.